Highly sensitive electrochemiluminescent sensing platform based on graphite carbon nitride nanosheets for detection of pyrophosphate ion in the synovial fluid

Accepted Manuscript Title: Highly sensitive electrochemiluminescent sensing platform based on graphite carbon nitride nanosheets for detection of Pyrophosphate Ion in the synovial fluid Author: Huifeng Xu Xi Zhu Yongqiang Dong Haishan Wu Yingmei Chen Yuwu Chi PII: DOI: Reference:

S0925-4005(16)30732-8 http://dx.doi.org/doi:10.1016/j.snb.2016.05.056 SNB 20215

To appear in:

Sensors and Actuators B

Received date: Revised date: Accepted date:

12-11-2015 6-5-2016 10-5-2016

Please cite this article as: Huifeng Xu, Xi Zhu, Yongqiang Dong, Haishan Wu, Yingmei Chen, Yuwu Chi, Highly sensitive electrochemiluminescent sensing platform based on graphite carbon nitride nanosheets for detection of Pyrophosphate Ion in the synovial fluid, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.05.056 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Highly sensitive electrochemiluminescent sensing platform based on graphite carbon nitride nanosheets for detection of Pyrophosphate Ion in the synovial fluid Huifeng Xua, b, Xi Zhuc, Yongqiang Donga, HaishanWua, Yingmei Chena, Yuwu Chia* a MOE Key Laboratory of Analysis and Detection for Food Safety, State Key Laboratory of Photo catalysis on Energy and Environment, and College of Chemistry, Fuzhou University, Fujian 350108, China. b Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350122, P. R. China c College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou,Fujian 350002, China.

Abstract Graphite carbon nitride nanosheets (g-C3N4 NSs) are the promising metal-free polymer-like

semiconductor

nanomaterials,

which

exhibit

excellent

electrochemiluminescence (ECL) behavior. The g-C3N4 NSs modified the glass carbon electrode showed the obvious ECL response using 10 mM S2O82- as the co-reactant at the scan rate of 0.1 V/s. The presence of Cu2+ would quench ECL emission due to the photo induced electron transfer (PET). Pyrophosphate anion (PPi),

*Corresponding

author; E-mail: [email protected]; Tel/Fax: +86-591-22866137.

1

an important anion in several bioenergetics and metabolic processes, can chelate with Cu2+ with a strong affinity. The introduction of PPi could release Cu2+ from Cu2+-g-C3N4 NSs system, resulting in the ECL recovery. The dosage of Cu2+ and the ECL recover time were further investigated. Under the optimized condition PPi can be detected in the range of 2.0 ~ 800 nM with the detection limit of 75 pM based on 3ơ/slope. This ECL sensor also possessed good selectivity to PPi, and has been used to detect PPi in the synovial fluid. Key

words:

Graphite

carbon

nitride

nanosheets

(g-C3N4

NSs);

electrochemiluminescent; copper ion; pyrophosphate anion (PPi); synovial fluid

1. Introduction Electrochemiluminescence (ECL) is a chemiluminescent reaction of species generated electrochemically at an electrode surface [1, 2]. ECL presents attractive advantages over the photoluminescence (PL) technique due to its low background emission, high sensitivity, and easy control[3]. It is well-known that Ru(bpy)32+, luminol and lucigenin are classical ECL luminophores[2, 4]. Besides, various inorganic quantum dots, like CdSe and CdS-CdSe, have been widely used as ECL luminophores[5-8]. However, these luminophores suffer from several disadvantages, such as expensive price, certain toxicity, poor chemically/thermally stability. Hence, the researchers try their best to explore more novel ECL luminophores. As a metal-free polymer-like semiconductor material, bulk graphite carbon nitride (g-C3N4) has been regarded as a valuable extension of carbon in material applications due to its special structure, optical and electrical properties[9]. Bulk 2

g-C3N4 has

attracted

considerable

attention

in

various

fields,

such

as

photo-catalysis[10], photo-degradation[11], photo electronic devices[12, 13], and chemical sensors[14-17] owing to its outstanding photoelectric performance and catalytic properties. However, the bulk g-C3N4 is not suitable for the application in analytical field due to its macroscopic size and poor solubility. To overcome these disadvantages, recently, ultrathin (single or several-layer) g-C3N4 nanosheets (g-C3N4 NSs) have been exfoliated from the bulk g-C3N4. Besides good water-solubility and atomically thick two-dimensional (2-D) structure, g-C3N4 NSs showed high quantum yields, strong ECL activity, high stability, and good biocompatibility. Therefore, g-C3N4 NSs have been applied in various fields, including

fluorescent[18-20],

photo

electrochemical

device[21-23],

and

bio-imaging[24]. In addition, ECL behavior of g-C3N4 NSs has also attracted growing interest due to its outstanding ECL activity. They have been used as the ECL luminophore for the detection of metal ions, and biomolecules [25-27]. Our group also has fabricated a series of ECL g-C3N4 NSs-based sensors for biosensing [26, 28-30]. Considering the promising potential of g-C3N4 NSs, more efforts are needed to deeply explore the application of g-C3N4 NSs in ECL sensing system. With the arrival of the aging society in the world, more and more attention is drawn to the aging related diseases. Specially, arthritis is one of the most frequent disorders directly correlated with age, which has already been a significant health care problem and aggravate the economic burden in the aging society. Therefore, monitoring the arthritis process is of great importance in the clinic diagnosis and 3

therapy of arthritic diseases. Pyrophosphate anion (P2O74-, PPi) is an important anion in biological systems, which plays significant roles in several bioenergetics and metabolic processes, especially the pathological processes of arthritis [31-36]. It has been also considered that PPi could be used as a potential biomarker for the clinicdiagnosis and therapy of arthritic diseases [32, 35, 37]. It is reported that high level of PPi in the synovial fluid has been found in the patients who suffered from calcium pyrophosphate dehydrates deposition diseases (CPDD) and from the diseases associated with CPDD, such as chondrocalcinosis and hypophosphatasis[32, 35]. In this regard, the monitoring of PPi is vital for clinic diagnosis and therapy of arthritic diseases. During the past decades, vast colorimetric and fluorescent sensors based on various organic probes are general approaches for the detection of PPi[38-48]. Additionally, nanomaterials have also been employed to fabricate the sensing platforms

for

assaying

PPi,

including

silica nanoparticles[49,

50],

gold

nanoparticles[51, 52], gold nanoclusters[53], graphene oxide, and magnetic nanoparticles[54, 55]. To our best knowledge, the ECL sensor for the detection of PPi is very scarce[56]. PPi could bind with Cu2+ to form complex of Cu2+/PPi with the strong affinity, where the Cu2+/PPi ratio is 1:2 (the stability constant (K) of the complex PPi-Cu2+-PPi : log KCu‑PPi=12.45)[57, 58]. In this work, on the basis of this fact, we developed a simple and ultra-sensitive method for PPi assay, and this sensor can be used to determinate PPi in the synovial fluid of arthritis patients.

2. Experimental section 4

2.1. Chemicals and Reagents Dicyanamide was purchased from Sigma. Cupric chloride (CuCl2·2H2O) and potassium persulfate (K2S2O8) were obtained from Fuchen Chemical Reagent Co. (Tianjin, China). Phosphate buffer solution (0.1 M, pH 7.0) containing 10 mM K2S2O8 was used throughout the ECL detection. All other reagents were of analytical reagent grade and used without further purification. All aqueous solutions were prepared with doubly distilled water. 2.2. Apparatus Electrochemical (EC) and ECL measurements were carried out on an C and ECL detection system (MPI-E, Remex Electronic Instrument Lt. Co., Xi’an, China) Lt. Co., Xi’an, China). Transmission electron microscopy (TEM) images were recorded on an electronic microscope (TecnaiG2 F20S-TWIN 200 kV). To investigate the optical properties of the g-C3N4 NSs aqueous solution, the photoluminescent spectra were recorded on a fluorescence spectrophotometer (Cary Eclipse, Varian). 2.3. Synthesis of g-C3N4 NSs The g-C3N4 NSs were prepared following the previously reported literature [26]. Firstly, the bulk g-C3N4 was synthesized. 3 g of dicyanamide was placed in a tube furnace (GSL 1400X, Kejing Materials Technology Lt. Co., Hefei, China) and heated at 600 °C for 2 h under air condition with a ramp of about 3 °C/min for both the heating and cooling processes. Then the pale yellow bulk g-C3N4 was obtained. Next, for the purpose of obtain g-C3N4 NSs, 100 mg of bulk g-C3N4 powder was ground well with a mortar and a pestle, followed by dispersed in 100 mL of water and 5

ultrasound for 16 h. Subsequently, the initial formed suspension was centrifuged at about 6000 rmp to remove the residual unexfoliated g-C3N4. Finally, the supernatant was collected and concentrated on a rotary evaporator at 60 °C under reduced pressure, resulting in a milk-like suspension. The mass concentration of the g-C3N4 NS suspension was calculated by weighing the power dried from a certain volume of the suspension. 2.4. The preparation and modification of the electrode Prior to modification, the glass carbon electrode (GCE, 2mm in diameter) was successively polished in turn with 0.3 and 0.05 μm alumina slurry, and then was ultrasonically cleaned in water for 5 min. 10 μL of the prepared g-C3N4 NSs suspension was dropped onto the cleaned GCE and dried at room temperature to obtain a g-C3N4 NSs-modified electrode. 2.5. ECL detection of PPi Here, a three-electrode system consisting of an Ag/AgCl (saturated KCl) electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode and g-C3N4 NSs-modified GCE electrode as the working electrode was applied. During the detection, a scanning potential with the window of −1.1 V ∼ 0 V was applied, and the voltage of the photomultiplier tube (PMT) was set at 800 V. 2.6. ECL assay During the ECL process, the three-electrode system was inserted into 2 mL of PBS (0.1 M, pH 7.0) containing 10 mM K2S2O8 and 100 nM Cu2+ in an ECL detector cell. For the quantitative analysis, standard PPi solutions with different concentrations 6

and sample solutions were introduced into above cell, and the maximum of ECL intensity of each potential scan was recorded.

3. Results and discussion 3.1. Characterization of g-C3N4 NSs The morphology and microstructure of g-C3N4 NSs were investigated with TEM (Fig.1A). It is clear that g-C3N4 NSs are lamellar and planar thin nanosheets in the TEM image. In addition, the morphology of g-C3N4 NSs modified Indium Tin Oxide (ITO) electrode was also observed by scanning electron microscopy (SEM). From Fig. 1B, it could be noticed the stacked lamellar texture of g-C3N4 NSs on the ITO electrode. These nearly transparent layers are also an evidence of the ultrathin thickness of the prepared g-C3N4 NSs. Under room light, g-C3N4 NSs suspension solution is milk-like (left, Fig. 1C). The diluted g-C3N4 NSs suspension shows bright blue luminescence when excited with a 365 nm UV light lamp (right, Fig. 1C). Its maximum PL excitation (Fig. 1D, curve a) and emission (Fig. 1D, curve b) wavelengths were 315 and 435 nm, respectively. These results were similar with those previously reported [26, 27], indicating the successful preparation of g-C3N4 NSs. 3.2. The characterization of the g-C3N4 NSs modified GCE The modified process of the electrode were examined by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) experiments in the buffer solution containing 5.0 mM Fe(CN)63-. According to Fig. 2A, the bare GCE shows a 7

small semicircle domain (curve a), indicating a low Ret value of the redox couple of [Fe(CN)6]3−/4−. An increasing semicircle in Ret values is observed through the immobilization of the g-C3N4 NSs, which may be attributed that the semiconductor nature of g-C3N4 may slow down the transfer rate of the electron in the GCE surface (curve b). CVs of different electrodes were also investigated (Fig.2B). Compared with the bare GCE, g-C3N4 NS modified GCE showed a slightly decreased peak current and increasing peak-to-peak separation, which is similar with the conclusion of EIS. From these results, it is proved that g-C3N4 NSs has been modified onto the GCE. 3.3. ECL behaviors of the g-C3N4 NSs modified GCE We investigated the ECL behaviors of the g-C3N4 on the GCE using S2O82- as the co-reactant. As shown in Scheme 1A, the g-C3N4 NSs modified GCE shows a very strong cathodic ECL emission at -1.07 V(curve a) due to strong high-energy annihilation between electrons (injected into the conduction band (CB) of g-C3N4 NSs by the anodic electrode) and holes (injected into the valence band (VB) of g-C3N4 NSs by the electrogenerated SO4•− from S2O82−). After the introduction of 100 nM Cu2+ into the electrochemical cell (curve b), the ECL intensity of g-C3N4 NSs significantly decreases, indicating that Cu2+ can quench the ECL emission effectively. This phenomenon may be explained by that the appropriate redox potential of Cu2+/Cu+, lying between CB and VB of g-C3N4 NSs, causes photo-induced electron to transfer from CB to Cu2+, and thus leads to the ECL quenching. It is reported that PPi has a strong affinity to Cu2+[14], based on which we speculated that the chelation between PPi and Cu2+ would recover the quenched ECL emission. Hence we added certain amount of PPi into the above solution to investigate the effect of PPi on the ECL intensity. The experimental result shows that the quenched ECL can be recovered obviously by the addition of PPi (curve c). It is worth noting that the ECL-CV curves have no obvious change after the addition of Cu2+ (curve b) and PPi(curve c), which 8

suggest that the addition of the determinand has little influence on the ECL process, but just leads to a simple quenching/ recovery of ECL intensity. The detailed sensing principle has been shown in Scheme 1B. Therefore, it is able to assay PPi using the g-C3N4 NSs modified GCE. 3.4. Optimal conditions for ECL emission CV and step potential are two commonly used methods in ECL detections. Although step potential can provide a faster detection, CV may provide more information about the ECL reactions during the detection. Therefore, CV are applied in the following ECL measurements. In order to achieve an ideal solid-state ECL emission of g-C3N4 NSs at GCE, we optimized several important experimental conditions, including the modified dosage of g-C3N4 NSs, the concentration of co-reactant K2S2O8, and the potential scan rate (Fig. 3). We used “relative ECL intensity” to stand for the ECL response, that is, the normalization of ECL intensity. It is defined as Ii/Imax, where Ii and Imax represent the ECL intensity and max value ECL intensity in the optimized condition of the condition optimization experiments, respectively. First, with increasing the concentration of g-C3N4 NSs modification solution, the ECL intensity increases gradually, and reaches a plateau at 1 mg/mL of g-C3N4 NSs (Fig. 3A). Thus, 1 mg/mL g-C3N4 NSs was selected. Second, the concentration of K2S2O8 was evaluated. As shown in Fig. 3B, ECL intensity enhanced with the increasing concentration of K2S2O8 and reaches a maximum value at 10 mM. Excessive S2O82(e.g. higher than 10 mM) may react readily with the negatively charged g-C3N4 and inhibit the formation of the excited-state g-C3N4*, leading to the decrease of ECL 9

intensity. Thus 10 mM was chosen as the optimal concentration of the co-reactant K2S2O8. Next, the influence of the potential scan rate on the ECL was investigated (Fig. 3C). The ECL intensity of the g-C3N4 NSs modified electrode has a maximum value when the potential scan rate is 0.10 V/s, thus, this scan rate was applied throughout the following experimental course. 3.5. ECL quenching by Cu2+ and its recovery by PPi As previously reported by the literature[14], Cu2+ can effectively quench the ECL of g-C3N4 by capturing the photo-excited electrons of g-C3N4. Therefore the quenching behavior of Cu2+ was investigated in detail. As shown in Fig. 4A, when added with 50 nM Cu2+, the ECL signal of g-C3N4 NSs declines rapidly with the time. After 5 min, the decrease of ECL signal is slight. Therefore, 5 min of quenching time was chosen. Subsequently, the effect of Cu2+ concentration in the solution on ECL was investigated. As shown in Fig. 4B, with increasing the concentration of Cu2+, the ECL signal decreases gradually. Until the concentration of Cu2+ exceeds 100 nM, the decrease becomes very slight. The reaction time of PPi with Cu2+ was also monitored. As shown in Fig. 4C, the ECL intensity gradually recovered upon the injection of 50 nM PPi into the reaction solution containing 100 nM Cu2+. After 30min, the ECL signal reaches a plateau. Therefore, 30 min was chosen as the recovery time. 3.6. ECL sensing of PPi Upon the optimal conditions, we constructed a facile “signal-on” ECL sensor for 10

PPi based on the g-C3N4 NSs modified GCE in the working solution containing 1 mM K2S2O8 and 100 nM Cu2+. In the absence of PPi, the sensor exhibited the weak ECL response due to the PET between Cu2+ and g-C3N4. While in the presence of PPi that can combine with Cu2+ to form the complex, the sensor gave a signal-on output. Moreover, with the increasing PPi concentration, the ECL response enhances gradually (Fig. 5A). When the rate of Cu2+/PPi was 1:2, ECL intensity recovered to 86.5%. ECL recovery reaches nearly 100% during the rate of 1:8. Fig. 5B shows that the ECL intensity is proportional to the logarithmic of PPi concentration in the range of 2.0 ~ 800 nM, with the regression equation shown as following: IECL=-312.85+1351.63 Log CPPi where IECL and CPPi stand for the ECL intensity and the concentration of PPi, respectively, and the co-efficient R2 is 0.9912. The limit of detection (LOD) of 75 pM was estimated based on 3/slope ( is the standard deviation of the blank sample measured 10 times). Table 1 indicates the sensing performance of different detection methods for PPi. It is obvious that our ECL sensor exhibits superior sensitivity to previously reported PPi sensors [44, 52-55, 59]. Moreover, as previously mentioned, g-C3N4 NSs solution shows bright blue luminescence at the UV light lamp, while the luminescence almost disappears in the presence of Cu2+ (the bottle a in Fig. 5(C)). In the addition of PPi, the luminescence increases gradually (bottle b to d), which is similar with the results from ECL. However, it is still difficult to distinguish these color changes as quantitative analysis, especially when the concentration of PPi is low to micromole level. It is also proved that this proposed ECL sensor has excellent 11

sensitivity toward the detection of PPi. 3.7. Stability and selectivity reproducibility Fig. 6A displays the signals of the g-C3N4 NSs-based ECL sensor under 6 cycles of continuous potential scans between -1.1 and 0 V. Very stable ECL signals were observed with the relative standard deviation (RSD) of 2.1%, indicating that the proposed ECL sensor has good stability. To examine the specificity of the fabricated ECL sensor, the influence of other anions on the g-C3N4 NSs-S2O82--Cu2+ ECL sensing system was evaluated. ECL intensities of sensing solutions are nearly unchanged and keep in low values upon the addition of 1 mM interfering anions such as Cl-, F-, Br-, I-, H2PO4-, SO42- and NO3-, whereas the addition of 50 nM PPi gives rise to a large ECL signal (Fig.6B). The results indicate that the selectivity of the ECL sensor was acceptable. We also prepared different batches of g-C3N4 NSs-based sensors. Similar results have been achieved, showing that this type of sensor has good reproducibility. 3.8. Assay of PPi in the synovial fluid Here, we applied this sensor for the determination of PPi in the synovial fluid of arthritis patients from Fuzhou Second Affiliated Hospital of Xiamen University. The selected synovial fluid was filtered by a filter membrane (0.2 m), and diluted to a certain volume. After the detection for three times, there is about 24.6 M PPi found in the synovial fluid with 5.7% of relative standard deviation (RSD), which is similar with the values reported in the literature[60, 61]. Furthermore, 28.6 M PPi was assayed using the competitive coordination of Cu2+ between Cysteine and PPi and the 12

optical property of Au-NPs from Mao’ group[51]. These results showed that this ECL can be detected PPi in the synovial fluid.

4. Conclusions In the present work, a simple and selective ECL sensor for the rapid, ultrasensitive detection of PPi has been developed based on the efficient ECL emission of g-C3N4 NSs. The developed ECL sensor shows high sensitivity, wide linear range, good reproducibility, and low detection limit (sub-picomole for PPi). Results indicates that g-C3N4 NSs hold huge promising potential as an alternative for traditional ECL luminophores not only for the highly ECL activity but also its nontoxic, inexpensive and cost-effective advantages.

Acknowledgement. This project was financially supported by NSFC (81202912, 21375020, 21305014), and the Natural Sciences Foundation of Fujian Province (2014J05013, 2016J01396), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20133514110001). H. Xu also thanks the University Distinguished Young Research Talent Training Program of Fujian Province.

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and production of calcium pyrophosphate dihydrate crystals, Annals of the Rheumatic Diseases, 42 (1983) 27-37. [32] M. Doherty, C. Belcher, M. Regan, A. Jones, J. Ledingham, Association between synovial fluid levels of inorganic pyrophosphate and short term radiographic outcome of knee osteoarthritis, Annals of the Rheumatic Diseases, 55 (1996) 432-436. [33] K. Johnson, R. Terkeltaub, Inorganic pyrophosphate (PPI) in pathologic calcification of articular cartilage, Frontiers in Bioscience, 10 (2005) 988-997. [34] L.M. Ryan, D.J. Mccarty, Understanding inorganic pyrophosphate metabolism: toward prevention of calcium pyrophosphate dihydrate crystal deposition, Annals of the Rheumatic Diseases, 54 (1995) 939–941. [35] R.A. Terkeltaub, Inorganic pyrophosphate generation and disposition in pathophysiology, American Journal of Physiology Cell Physiology, 281 (2001) C1-C11. [36] R.C. Lawrence, C.G. Helmick, F.C. Arnett, R.A. Deyo, D.T. Felson, E.H. Giannini, S.P. Heyse, R. Hirsch, M.C. Hochberg, G.G. Hunder, M.H. Liang, S.R. Pillemer, V.D. Steen, F. Wolfe, Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States, Arthritis & Rheumatism, 41 (1998) 778-799. [37] A. Micheli, J. Po, G.H. Fallet, Measurement of soluble pyrophosphate in plasma and synovial fluid of patients with various rheumatic diseases, Scandinavian journal of rheumatology, 10 (1981) 237-240. [38] H.L. Dong, J.H. Im, S.U. Son, Y.K. Chung, J.I. Hong, An Azophenol-based Chromogenic Pyrophosphate Sensor in Water, Journal of the American Chemical Society, 125 (2003) 7752-7753. [39] S. Mizukami, T. Nagano, Y. Urano, A. Odani, K. Kikuchi, A fluorescent anion sensor that works in neutral aqueous solution for bioanalytical application, Journal of the American Chemical Society, 124 (2002) 3920-3925. [40] D.H. Lee, S.Y. Kim, J.I. Hong, A Fluorescent Pyrophosphate Sensor with High Selectivity over ATP in Water, Angewandte Chemie International Edition, 116 (2004) 4881-4884. [41] H.N. Lee, K.M.K. Swamy, S.K. Kim, J.-Y. Kwon, Y. Kim, S.-J. Kim, Y.J. Yoon, J. Yoon, Simple but Effective Way to Sense Pyrophosphate and Inorganic Phosphate by Fluorescence Changes, Organic Letters, 9 (2006) 243-246. [42] H.N. Lee, Z. Xu, S.K. Kim, K.M. Swamy, Y. Kim, S.J. Kim, J. Yoon, Pyrophosphate-Selective Fluorescent Chemosensor at Physiological pH:65 Formation of a Unique Excimer upon Addition of Pyrophosphate, Journal of the American Chemical Society, 129 (2007) 3828-3829. [43] S. Bhowmik, B.N. Ghosh, V. Marjom01ki, K. Rissanen, Nanomolar pyrophosphate detection in water and in a self-assembled hydrogel of a simple terpyridine-Zn2+ complex, Journal of the American Chemical Society, 136 (2014) 5543-5546. [44] R. Villamil-Ramos, Y. AK, Selective fluorometric detection of pyrophosphate by interaction with alizarin red S-dimethyltin(IV) complex, Chemical Communications, 47 (2011) 2694-2696. [45] S.K. Kim, N.J. Singh, J. Kwon, I.C. Hwang, J.P. Su, K.S. Kim, J. Yoon, Fluorescent imidazolium receptors for the recognition of pyrophosphate, Tetrahedron, 62 (2006) 6065–6072. [46] S.K. Kim, D.H. Lee, J.I. Hong, J. Yoon, Chemosensors for pyrophosphate, Accounts of Chemical Research, 42 (2008) 23-31. [47] S. Yang, G. Feng, N.H. Williams, Highly selective colorimetric sensing pyrophosphate in water by a NBD-phenoxo-bridged dinuclear Zn(II) complex, Organic & Biomolecular Chemistry, 10 (2012) 5606-5612. [48] X. Huang, Z. Guo, W. Zhu, Y. Xie, T. H, A colorimetric and fluorescent turn-on sensor for pyrophosphate anion based on a dicyanomethylene-4H-chromene framework, Chemical Communications, 41 (2008) 5143-5145. [49] D.J. Oh, K.M. Kim, K.H. Ahn, Nanoparticle-based Indicator-Displacement Assay for Pyrophosphate, Chemistry – An Asian Journal, 6 (2011) 2034-2039. [50] J.F. Zhang, M. Park, W.X. Ren, Y. Kim, S.J. Kim, J.H. Jung, J.S. Kim, A pellet-type optical nanomaterial of silica-based naphthalimide-DPA-Cu(ii) complexes: recyclable fluorescence detection of pyrophosphate, Chemical Communications, 47 (2011) 3568-3570. [51] J. Deng, P. Yu, L. Yang, L. Mao, Competitive Coordination of Cu2+ between Cysteine 16

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17

Huifeng Xu received his MS degree in 2010 from Fuzhou University, China. She is working as a research assistant in Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, China, and currently a PhD student in College of Chemistry, Fuzhou University. Her research interests focus on functional 2D nanosized materials and their sensing application. Xi Zhu received his PhD degree in 2012 from Fuzhou University, China. Currently, he is working as an associate professor in College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou Haishan Wu obtained his BS degree in 2015 from Fuzhou University, China. He is currently working as a graduate student in College of Chemistry, Fuzhou University, Fuzhou. Yingmei Chen received her BS degreein 2008 from Fuzhou University, China. She is currently studying for an MD-PhD degree in College of analytical chemistry, Fuzhou University, Fuzhou. Yongqiang Dong received his BS (2006) and PhD (2011) degrees from the department of chemistry, Fuzhou University, China. He entered Nanyang Technologic University, Singapore as a postdoctoral researcher from 2011 to 2013. Currently, he is an associate professor of chemistry at Fuzhou University. His research interests mainly relate to the synthesis, luminescent properties and sensing applications of carbon based dots. Yuwu Chi received his BS (1992) and MS (2000, Prof. Guonan Chen) degrees in Analytical Chemistry at Fuzhou University, and then a Ph D degree in applied physics (2005, Prof. Koichi Aoki) from the University of Fukui. From 1992 to 2002, he worked at Fuzhou University as a research assistant and a lecturer in turn. In 2005, he re-entered Fuzhou University after obtaining his Ph D degree in Japan, and worked as a professor at the College of Chemistry. Presently, his research interests focus on functional carbon materials, electrochemiluminescence, electroanalysis, chemical and biochemical sensing.

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Figure 1. (A) TEM and (B) SEM image of g-C3N4 NSs. (C) The photograph of g-C3N4 NSs under room light (left) and UV light (right). (D) Excitation spectrum (curve a), and emission spectrum (curve b) of g-C3N4 NSs.

19

a

-300

b

Current (10-5A)

A

-250

Z"(k)

-200 -150 -100 -50 0 0

100

200

300

400

500

600

8

a

B

0 -4 -8 0.6

Z'(k)

b

4

0.4

0.2

0.0

-0.2

Potential (V)

Figure 2. (A) Electrochemical impedance spectroscopy and (B) cyclic voltammetry of different GCEs in the solution containing [Fe(CN)6]3−/4−,(a) bare GCE, (b) g-C3N4 NS-modified GCE.

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0.8

A

0.6 0.4 0.2 0.0

Relative ECL intensity

0.0 0.5 1.0 1.5 2.0 Concentration of g-C3N4(mg/ml)

1.0

Relative ECL intensity

Relative ECL intensity

1.0

1.0

B

0.8 0.6 0.4 0.2 0.0 0.0

0.5

1.0

1.5

2.0

Concentration of K2S2O8 ( mM)

C

0.8 0.6 0.4 0.0

0.1

0.2

Scan rate ( V /s)

Figure 3. Effects of experimental conditions on the ECL response of g-C3N4 NSs-modified electrode: A) the dosage of g-C3N4 NSs; B) the concentration of co-reactant K2S2O8; C) the potential scan rate.

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A

0.8 0.6 0.4 0.2 0.0 0

1

2 3 4 5 Time (min)

Relative ECL intensity

Relative ECL intensity Relative ECL intensity

1.0

1.0

B

0.8 0.6 0.4 0.2 0.0

1.0

C

0.8 0.6 0.4 0.2 0.0 0

0 40 80 120 160 2+ Concentration of Cu (nM)

6

10

20

30

40

Time (min)

Figure 4. A) The time evolution of the ECL response in the presence of 50 nM Cu2+. B) The dependence of the ECL intensity on the Cu2+ concentrations. C) The recovery of the ECL response in presence of 100 nM Cu2+ and 50 nM PPi.

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Figure 5. A) ECL curves upon the introduction of different PPi concentrations into the sensing system. Curves (a) to (i): 0, 2, 5, 15, 50, 100, 200, 400, and 800 nM. B) The plot of the ECL intensity versus the logarithmic of PPi concentration. C) The photograph of different g-C3N4 NSs solutions under UV light (right). The bottles from a to d represent g-C3N4 NSs solutions containing 1mM Cu2+, 1mM Cu2+ and 1mM PPi, 1mM Cu2+ and 2mM PPi, and 1mM Cu2+ and 8mM PPi, respectively.

23

A

ECL intensity (a.u.)

300 250 200 150 100 50 0 0

20

40

60

80

100

120

ECL intensity (a.u.)

Time (s)

B

1750 1400 1050 700 350 0

PPi Cl-

F

Br

-

I

-

2-

Pi SO4 NO3

-

Figure 6. A) The stability of ECL sensor. B) The selectivity of the sensor upon exposure to various anions. The concentration of PPi was 50 nM and the concentrations of other anions were all 1 mM.

24

ECL intensity (a.u.)

A

4000

a

3000 2000 1000 0

c b

-1.2 -1.0 -0.8 -0.6 -0.4 -0.2

0.0

Potential (V)

Scheme 1. A) ECL responses of the g-C3N4 NSs-modified GCE under different conditions: (a) S2O82-; (b) S2O82- + Cu2+; (c) S2O82- + Cu2+ +PPi. B) The sensing principle for PPi.

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Table 1 The sensing performance of different detection methods for PPi Probe

Detection method

Detection limit

Ref.

Fluorescent

3 M

44

Gold nanoparticle

Colorimetric

200 nM

52

BSA-protected gold nanoclusters

Fluorescent

0.083 M

53

Fluorescent

7 nM

54

Fluorescent

76 nM

55

Fluorescent

1 M

59

Dimethyltin(IV)–alizarin red S complex

boron dipyrromethene-conjugated adenosine 5'-triphosphate FAM labeled single-stranded DNA

2,2’-Biimidazole-Based Conjugated Polymer This g-C3N4 NSs

ECL

75 pM work

26