Biosensors and Bioelectronics 63 (2015) 478–482
Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Electrogenerated chemiluminescence behavior of peptide nanovesicle and its application in sensing dopamine Chunxiu Huang a, Xu Chen a,n, Yanluo Lu a, Hui Yang b, Wensheng Yang a,n a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Degeneration and Repairing, Key Laboratory for Neurodegenerative Diseases of Ministry of Education, Beijing 100069, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 July 2014 Accepted 19 July 2014 Available online 30 July 2014
The electrogenerated chemiluminescence (ECL) behavior of the bioinspired peptide nanovesicles (PNVs) was reported for the first time. The PNVs modified glassy carbon electrodes have shown a stable and efficient cathodic ECL signal with K2S2O8 as coreactant in aqueous solution. The possible ECL reaction mechanism was proposed. Dopamine (DA) was chosen as a model analyte to study the potential of the PNVs in the ECL analytical application. It was found that the ECL intensity of the PNVs was effectively increased by trace amounts of DA. The limit of detection was estimated to be 3.15 pM (S/N ¼3). These results suggest that the PNVs could be a new class of promising materials for the ECL design and bioassays in the future due to their fascinating features, such as excellent biocompatibility, tunable composition as well as capability of molecular recognition. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Peptide nanovesicles Dopamine
1. Introduction Electrogenerated chemiluminescence (ECL) is an attractive analytical technique due to its high sensitivity and wide linear range. In recent years, the employment of nanomaterials has promoted the rapid development of ECL in bioanalytical applications (Ding et al., 2002; Miao, 2008; Li et al., 2012). Semiconductor nanocrystals, such as CdSe (Myung et al., 2003) and CdTe (Cheng et al., 2010), are often used in ECL studies. However, the inherent toxicity from the heavy metal elements limits their wide applications in the bioassay. To maintain a benign environment, lowtoxicity silicon (Ding et al., 2002) and carbon (Zheng et al., 2009) nanocrystals (NCs) as well as Au nanoclusters (Li et al., 2011) have been tried in ECL reactions. Nevertheless, the analytical performances based on their ECL still needed to be further improved. Therefore, the development of new highly efficient, biocompatible and tunable ECL nanoemitters is highly desirable for both fundamental and bioanalytical applications (Hu and Xu, 2010; Deng and Ju, 2013). Bioinspired nanomaterials, especially fabricated from peptide building blocks, are of increasing interest owing to their biocompatibility, well-defined structures and capability of molecular recognition (Hartgerink et al., 2001; Scanlon and Aggeli, 2008). Recently the simplest peptide building blocks of diphenylalanine n
Corresponding authors. Tel.: þ 86 10 64435271; fax: þ86 10 64425385. E-mail address: [email protected] (X. Chen).
http://dx.doi.org/10.1016/j.bios.2014.07.060 0956-5663/& 2014 Elsevier B.V. All rights reserved.
(FF), found as the structural motif for the β-amyloid associated with Alzheimer's disease, have attracted considerable attention (Reches and Gazit, 2003; Kim et al., 2010). Self-assembled FFbased nanostructures, especially peptide nanotubes (PNTs), have extraordinary mechanical strength and good chemical and thermal stability (Kol et al., 2005), which make them appealing structural elements for various applications (Yemini et al., 2005; Yan et al., 2010). Very recently, Rosenman et al. observed the quantum confinement (QC) phenomenon in PNTs (Amdursky et al., 2009a), peptide nanospheres (Amdursky et al., 2009b) and hydrogels (Amdursky et al., 2010a), which was found previously only in semiconductor crystals. Further investigation on these self-assembled peptide structures indicated that they were composed of nanocrystalline regions or quantum dots (QDs) (Amdursky et al., 2010b). These bionanostructures with QC features have exceptional electronic and photonic properties, which make them new candidates of environmentally clean optical materials for luminescence devices (Ryu et al., 2009; Yan et al., 2011; Kim et al., 2011). However, to the best of our knowledge, no research has been reported involving the FF and its derivate-based nanostructures in the ECL study. In the present work, we at the first time explored the ECL properties of cationic dipeptide (H-Phe-Phe-NH2 HCl, derived from FF, see Fig. 1A) self-assembled nanovesicles (PNVs). The nanospherical morphology is preferred in biosensing, labeling of biomolecules and bioimaging applications relative to long nanotubes. The cathodic ECL of the PNVs modified glassy carbon electrodes (GCE) was firstly observed in the presence of coreactant
C. Huang et al. / Biosensors and Bioelectronics 63 (2015) 478–482
479
Fig. 1. (A) Schematic illustration of cationic dipeptide self-assembly into PNVs. Inset: Molecular structure of cationic dipeptide. (B) TEM image of PNVs.
K2S2O8. The possible ECL reaction mechanism was proposed. The analytical application of the PNVs ECL was also demonstrated.
2. Experimental 2.1. Materials The cationic dipeptide (H-Phe-Phe-NH2 HCl, CDP) in a lyophilized powder was obtained from Bachem AG (Switzerland). 3-Hydroxytyramine hydrochloride (Dopamine) and 1.1.1.3.3.3-hexafluoro-2-propanol (HFIP) were purchased from Sigma-Aldrich. Phosphate-buffered solution (PBS) (pH 7.2, 0.2 M) was prepared using 0.2 M Na2HPO4, 0.2 M NaH2PO4 and 0.1 M KCl. All other reagents were of analytical grade and used as received without further purification. Ultrapure water was used throughout the experiments. 2.2. Apparatus Transmission electron microscopy (TEM) images were taken on a JEOL JEM-2100 electron microscope with an accelerating voltage of 100 kV. The ECL spectra were recorded by collecting the ECL data during the cyclic potential sweep with a series of optical filters. Cyclic voltammetric and ECL measurements were carried out with a Model MPI-E Electrochemiluminescence Analyzer (Xi’an Remex Analytic Instrument Co., Ltd., Xi’an, China). Detection was carried out in a static ECL system. The voltage of the photomultiplier tube (PMT) was set at 800 V and the scan rate was 100 mV s 1 in the process of ECL detection. A conventional three-electrode system was used in the experiment with a bare or modified glassy carbon working electrode (diameter 3 mm), a Pt wire counter electrode, and an Ag/AgCl reference electrode. 2.3. Synthesis of peptide nanovesicles (PNVs) PNVs were prepared according to a previously reported method (Yan et al., 2007). Fresh stock solutions were prepared by dissolving 5 mg lyophilized CDP powder in 40 μL HFIP. Then the stock solution was diluted to 10 mg mL 1 by using a pH 7.2 PBS. Finally, the 10 mg mL 1 solution was diluted to 1 mg mL 1 by using ultrapure water and the self-assembled PNVs were formed. To eliminate the possible residual monomer and cytotoxic HFIP, the PNVs solution was further dialyzed for 48 h with ultrapure water. The resulting solution was used in the experiments to the characterization of the PNVs and the fabrication of the modified electrodes.
2.4. Preparation of PNVs modified electrode Prior to use, the GCE was polished carefully with 1.0, 0.3 and 0.05 mm alumina slurry, followed by washing thoroughly with water. Then the electrode was sonicated successively in water and allowed to dry in a stream of N2. PNVs solution (optimized 6 mL) was dropped on the surface of the GCE and dried at room temperature.
3. Results and discussion 3.1. Characterization of PNVs Fig. 1B displays a TEM image of the PNVs. A typical spherical and vesicle structure with the diameters of 10 25 nm was observed. A typical atomic force microscopy (AFM) image (Fig. S1) of the PNVs on mica slice further confirmed the morphology of the PNVs. The cross-sectional profile shows that the PNVs are approximately 5 nm in height, indicating that they are flat (the diameter of the PNVs in two dimensions, 10 25 nm). Moreover, UV–vis spectrum of the PNVs solution showed the identical spikelike behavior (Fig. S2), indicating the existence of identical nanosize regions of QDs in the PNV structures (Amdursky et al., 2010b). Additionally, the photoluminescence (PL) spectrum of the PNVs solution exhibited two peaks under excitation at 250 nm (Fig. S3). The first peak was located at 281 nm, which is characterization of the phenylalanine residue (Mihalyi, 1968). A second peak was found at about 559 nm, which might be attributed to the assembled nanostructures (Amdursky et al., 2009a). 3.2. ECL behavior of PNVs modified electrode Fig. 2A shows the ECL responses obtained on the bare and PNVs modified GCEs in 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl. Herein, K2S2O8 played a coreactant role, which is generally used in ECL systems. It was found that only a weak ECL signal (line a in Fig. 2A) was observed on the bare GCE when the potential was cycled between 0 and 1.60 V. However, a strong ECL signal, about 7 times than that of the bare GCE, was obtained at 1.30 V on the PNVs modified GCE (line b in Fig. 2A). These results suggested that the ECL signal originated from the system of PNVs. Furthermore, the ECL signal remained at an almost constant value during consecutive cyclic potential scanning (Fig. 2B), indicating the good reversibility of the modified electrode. Additionally, the ECL working conditions were optimized and shown in Fig. S4. The optimal parameters were used throughout this work. The ECL spectra were also measured by employing a series of optical filters, and a distinguished ECL spectrum peak at approximately 627 nm was observed (Fig. 2C). It was different with that
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Fig. 2. (A) ECL responses obtained on a bare (a) and PNVs modified (b) GCEs. (B) Time-dependent ECL signals of the PNVs modified GCE. (C) ECL spectrum of PNVs. All ECL signals were collected in 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl.
obtained on the bare GCE in the same condition (Fig. S5), suggesting that the PNVs played a key role in the ECL mechanism. In addition, compared to the PL emission spectrum peak of the PNVs at 559 nm, there was a significant spectral shift between the ECL and PL. The red shift was also observed in the previously reported ECL of Si (Ding et al., 2002) and CdSe NCs (Myung et al., 2003), which was explained to the strong role of surface states on the ECL process of the NCs. This indicated that the prepared PNVs had a sensitive surface state. In order to explore the ECL mechanism of the PNVs, the effect of various conditions on the ECL behavior of PNVs modified GCEs was further investigated. When K2S2O8 was absent in reaction solutions, no appreciable ECL signal was observed (Fig. S6), indicating that S2O82 played an important role in the ECL process as a coreactant. Moreover, the PNVs modified GCE was also scanned between þ2.0 and 1.6 V. The anodic ECL response was not found, and the cathodic ECL signal was basically consistent with that obtained between 0 and 1.6 V (Fig. S7). This suggested the coreactant ECL contributed to the whole ECL dominantly. In addition, the ECL response was not obviously changed in oxygen-depleted conditions (not shown), revealing that the ECL buildup was not primarily on the presence of oxygen. All of the above ECL properties indicated that the PNVs ECL was coreactant mechanism. In all coreactant ECL system toward the light emission, four processes generally are involved, namely (a) redox reactions at
electrode, (b) homogenous chemical reactions, (c) excited state species formation and (d) light emission (Miao, 2008). Herein, when the potential is swept to a sufficiently negative potential, an electron from the working electrode might be injected to PNVs to generate the PNVsd (Fig. S8). Meanwhile, the coreactant S2O82 could also be reduced during this cathodic process to SO4d and SO42 on the PNVs modified GCE (Fig. S9). The formed intermediate SO4d would subsequently react with PNVsd to produce the excited state PNVs* via electron transfer from PNVsd to SO4d . Another possibility is that the strongly oxidizing species SO4d might oxidize PNVs to PNVsd þ , which is analogous to the formation of aromatic hydrocarbons radical cation upon oxidation by SO4d (Fabrizio et al., 2000). Subsequently, the annihilation of PNVsd þ and PNVsd ions generates the excited sate PNVs* and PNVs. Finally, PNVs* returns to the ground state accompanied with photon irradiation, thus the ECL emission could be obtained. The possible ECL processes of the PNVs are described with the following equations: PNVsþ e -PNVsd
(1)
S2 O82 − + e− → SO42 − + SO4⋅−
(2)
PNVs⋅− + SO4⋅− → PNVs⁎ + SO42 −
(3)
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Fig. 3. (A) ECL profiles of the PNVs modified GCE in the presence of different DA concentrations in 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl. (B) Linear calibration plot for DA detection.
and/or
PNVs + SO4⋅− → PNVs+ + SO42 −
(4)
PNVs þ þPNVsd -PNVs* þPNVs
(5)
finally, PNVsn-PNVs þhν
(6)
3.3. Application of ECL from PNVs To demonstrate the sensing performance of the PNVs modified GCE, dopamine (DA, an important neurotransmitter) was chosen as a model analyte. When a trace of DA was injected into the 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl, the ECL emission showed an increase (Fig. 3A). Because the surface of the PNVs consist of aromatic stacking arrangements (Yan et al., 2010), so DA is easy to be adsorbed on the PNVs due to strong π–π interaction. The adsorption of DA on the PNVs surface could accelerate the electron transfer (Wang, Y., et al., 2009) from the DA directly to the PNVs, which finally might lead to the enhancement of ECL signal (Yuan et al., 2014). The improved current response could be obtained at the PNVs modified electrode in the presence of DA (Fig. S10), indicating that DA could indeed facilitate the electrochemical behavior in this system. With increasing the concentration of DA, the ECL intensity increased linearly from 10 to 200 pM (n ¼10, R¼0.9998). When further increasing the concentration of DA, the ECL response reached to a saturated value. The corresponding calibration curve (Fig. 3B) could be well fitted with the following equation:
I /I0 = 1.0003 + 0.0098 × CDA (pM)
(7)
where I0 is the initial ECL intensity, I is the ECL intensity at a given concentration of DA. The detection limit was estimated to be 3.15 pM for DA at a signal/noise ratio of 3, which is much lower than those of some inorganic NCs such as CdSe (0.5 mM) (Liu et al., 2008) and Ag2Se (0.1 mM) (Cui et al., 2012), as well as Au (∼1.0 mM) (Hu and Xu, 2010) and Ag (0.92 nM) (Liu et al., 2013) nanoclusters. These results indicate that the prepared PNVs modified GCE could be used for the monitoring of trace amounts of DA.
An interference investigation was also performed by using the solution containing 100 pM DA and 200 pM common co-existing substances such as uric acid, ascorbic acid, glucose, sucrose, urea, cysteine, catechol, acetone, Na þ and Cu2 þ , respectively. No significant change (less than 71%) was observed. Furthermore, increasing the concentrations of interference reagents to be 0.5 mM, the interferences were less than 73%. The good selectivity for DA might be attributed to the efficient electron transfer between DA and PNVs (Li et al., 2011, Wang, G.L., et al., 2009). In addition, five PNVs modified GCEs prepared under the same conditions showed an acceptable reproducibility with a relative standard deviation (RSD) of 3.14% for the detection of 100 pM DA. The prepared PNVs modified GCE maintained 96.1% of the original response after storage in dark and 4 °C refrigerator for 4 weeks, indicating the good stability of the sensor. In addition, the practical drug (dopamine hydrochloride injection) was also measured based on the PNVs modified electrode. After diluting the injection solution, the concentration of DA in the solution was detected to be 19.89 70.25 pM by ECL, close to the given dilute solution value of 20 pM. The relative standard deviation (RSD) for five measurements was 1.43%. These results indicate that this method has acceptable selectivity and accuracy.
4. Conclusions In conclusion, ECL was first observed from the PNVs derived from dipeptide building blocks. The ECL mechanism of the PNVs was possibly ascribed to cathodic coreactant mechanism. The highly sensitive detection for the model analyte DA has been obtained based on increasing ECL intensity of the PNVs modified GCE. Although the ECL intensity of the PNVs modified GCE was not very high at present, preliminary experimental results are promising, indicating that PNVs could be a new class of material for ECL sensing. More importantly, the present study opens up a new avenue to design and development of ECL devices from bioinspired materials. The variety of amino acids and formed diverse nanostructures would provide the endless combinatorial options for these biomaterials. Finally, we believe that the use of the bioinspired nanomaterials with the inherent biocompatibility and capability of molecular recognition would bring huge potential in the ECL bioanalytical applications.
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Acknowledgments This work was supported by the National Basic Research Program of China (No. 2011CBA00508) and the National Natural Science Foundation of China (No. 21175009).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.060.
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Contents lists available at ScienceDirect
Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
Short communication
Electrogenerated chemiluminescence behavior of peptide nanovesicle and its application in sensing dopamine Chunxiu Huang a, Xu Chen a,n, Yanluo Lu a, Hui Yang b, Wensheng Yang a,n a
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China Beijing Institute for Neuroscience, Capital Medical University, Beijing Center of Neural Degeneration and Repairing, Key Laboratory for Neurodegenerative Diseases of Ministry of Education, Beijing 100069, China
b
art ic l e i nf o
a b s t r a c t
Article history: Received 9 July 2014 Accepted 19 July 2014 Available online 30 July 2014
The electrogenerated chemiluminescence (ECL) behavior of the bioinspired peptide nanovesicles (PNVs) was reported for the first time. The PNVs modified glassy carbon electrodes have shown a stable and efficient cathodic ECL signal with K2S2O8 as coreactant in aqueous solution. The possible ECL reaction mechanism was proposed. Dopamine (DA) was chosen as a model analyte to study the potential of the PNVs in the ECL analytical application. It was found that the ECL intensity of the PNVs was effectively increased by trace amounts of DA. The limit of detection was estimated to be 3.15 pM (S/N ¼3). These results suggest that the PNVs could be a new class of promising materials for the ECL design and bioassays in the future due to their fascinating features, such as excellent biocompatibility, tunable composition as well as capability of molecular recognition. & 2014 Elsevier B.V. All rights reserved.
Keywords: Electrochemiluminescence Peptide nanovesicles Dopamine
1. Introduction Electrogenerated chemiluminescence (ECL) is an attractive analytical technique due to its high sensitivity and wide linear range. In recent years, the employment of nanomaterials has promoted the rapid development of ECL in bioanalytical applications (Ding et al., 2002; Miao, 2008; Li et al., 2012). Semiconductor nanocrystals, such as CdSe (Myung et al., 2003) and CdTe (Cheng et al., 2010), are often used in ECL studies. However, the inherent toxicity from the heavy metal elements limits their wide applications in the bioassay. To maintain a benign environment, lowtoxicity silicon (Ding et al., 2002) and carbon (Zheng et al., 2009) nanocrystals (NCs) as well as Au nanoclusters (Li et al., 2011) have been tried in ECL reactions. Nevertheless, the analytical performances based on their ECL still needed to be further improved. Therefore, the development of new highly efficient, biocompatible and tunable ECL nanoemitters is highly desirable for both fundamental and bioanalytical applications (Hu and Xu, 2010; Deng and Ju, 2013). Bioinspired nanomaterials, especially fabricated from peptide building blocks, are of increasing interest owing to their biocompatibility, well-defined structures and capability of molecular recognition (Hartgerink et al., 2001; Scanlon and Aggeli, 2008). Recently the simplest peptide building blocks of diphenylalanine n
Corresponding authors. Tel.: þ 86 10 64435271; fax: þ86 10 64425385. E-mail address: [email protected] (X. Chen).
http://dx.doi.org/10.1016/j.bios.2014.07.060 0956-5663/& 2014 Elsevier B.V. All rights reserved.
(FF), found as the structural motif for the β-amyloid associated with Alzheimer's disease, have attracted considerable attention (Reches and Gazit, 2003; Kim et al., 2010). Self-assembled FFbased nanostructures, especially peptide nanotubes (PNTs), have extraordinary mechanical strength and good chemical and thermal stability (Kol et al., 2005), which make them appealing structural elements for various applications (Yemini et al., 2005; Yan et al., 2010). Very recently, Rosenman et al. observed the quantum confinement (QC) phenomenon in PNTs (Amdursky et al., 2009a), peptide nanospheres (Amdursky et al., 2009b) and hydrogels (Amdursky et al., 2010a), which was found previously only in semiconductor crystals. Further investigation on these self-assembled peptide structures indicated that they were composed of nanocrystalline regions or quantum dots (QDs) (Amdursky et al., 2010b). These bionanostructures with QC features have exceptional electronic and photonic properties, which make them new candidates of environmentally clean optical materials for luminescence devices (Ryu et al., 2009; Yan et al., 2011; Kim et al., 2011). However, to the best of our knowledge, no research has been reported involving the FF and its derivate-based nanostructures in the ECL study. In the present work, we at the first time explored the ECL properties of cationic dipeptide (H-Phe-Phe-NH2 HCl, derived from FF, see Fig. 1A) self-assembled nanovesicles (PNVs). The nanospherical morphology is preferred in biosensing, labeling of biomolecules and bioimaging applications relative to long nanotubes. The cathodic ECL of the PNVs modified glassy carbon electrodes (GCE) was firstly observed in the presence of coreactant
C. Huang et al. / Biosensors and Bioelectronics 63 (2015) 478–482
479
Fig. 1. (A) Schematic illustration of cationic dipeptide self-assembly into PNVs. Inset: Molecular structure of cationic dipeptide. (B) TEM image of PNVs.
K2S2O8. The possible ECL reaction mechanism was proposed. The analytical application of the PNVs ECL was also demonstrated.
2. Experimental 2.1. Materials The cationic dipeptide (H-Phe-Phe-NH2 HCl, CDP) in a lyophilized powder was obtained from Bachem AG (Switzerland). 3-Hydroxytyramine hydrochloride (Dopamine) and 1.1.1.3.3.3-hexafluoro-2-propanol (HFIP) were purchased from Sigma-Aldrich. Phosphate-buffered solution (PBS) (pH 7.2, 0.2 M) was prepared using 0.2 M Na2HPO4, 0.2 M NaH2PO4 and 0.1 M KCl. All other reagents were of analytical grade and used as received without further purification. Ultrapure water was used throughout the experiments. 2.2. Apparatus Transmission electron microscopy (TEM) images were taken on a JEOL JEM-2100 electron microscope with an accelerating voltage of 100 kV. The ECL spectra were recorded by collecting the ECL data during the cyclic potential sweep with a series of optical filters. Cyclic voltammetric and ECL measurements were carried out with a Model MPI-E Electrochemiluminescence Analyzer (Xi’an Remex Analytic Instrument Co., Ltd., Xi’an, China). Detection was carried out in a static ECL system. The voltage of the photomultiplier tube (PMT) was set at 800 V and the scan rate was 100 mV s 1 in the process of ECL detection. A conventional three-electrode system was used in the experiment with a bare or modified glassy carbon working electrode (diameter 3 mm), a Pt wire counter electrode, and an Ag/AgCl reference electrode. 2.3. Synthesis of peptide nanovesicles (PNVs) PNVs were prepared according to a previously reported method (Yan et al., 2007). Fresh stock solutions were prepared by dissolving 5 mg lyophilized CDP powder in 40 μL HFIP. Then the stock solution was diluted to 10 mg mL 1 by using a pH 7.2 PBS. Finally, the 10 mg mL 1 solution was diluted to 1 mg mL 1 by using ultrapure water and the self-assembled PNVs were formed. To eliminate the possible residual monomer and cytotoxic HFIP, the PNVs solution was further dialyzed for 48 h with ultrapure water. The resulting solution was used in the experiments to the characterization of the PNVs and the fabrication of the modified electrodes.
2.4. Preparation of PNVs modified electrode Prior to use, the GCE was polished carefully with 1.0, 0.3 and 0.05 mm alumina slurry, followed by washing thoroughly with water. Then the electrode was sonicated successively in water and allowed to dry in a stream of N2. PNVs solution (optimized 6 mL) was dropped on the surface of the GCE and dried at room temperature.
3. Results and discussion 3.1. Characterization of PNVs Fig. 1B displays a TEM image of the PNVs. A typical spherical and vesicle structure with the diameters of 10 25 nm was observed. A typical atomic force microscopy (AFM) image (Fig. S1) of the PNVs on mica slice further confirmed the morphology of the PNVs. The cross-sectional profile shows that the PNVs are approximately 5 nm in height, indicating that they are flat (the diameter of the PNVs in two dimensions, 10 25 nm). Moreover, UV–vis spectrum of the PNVs solution showed the identical spikelike behavior (Fig. S2), indicating the existence of identical nanosize regions of QDs in the PNV structures (Amdursky et al., 2010b). Additionally, the photoluminescence (PL) spectrum of the PNVs solution exhibited two peaks under excitation at 250 nm (Fig. S3). The first peak was located at 281 nm, which is characterization of the phenylalanine residue (Mihalyi, 1968). A second peak was found at about 559 nm, which might be attributed to the assembled nanostructures (Amdursky et al., 2009a). 3.2. ECL behavior of PNVs modified electrode Fig. 2A shows the ECL responses obtained on the bare and PNVs modified GCEs in 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl. Herein, K2S2O8 played a coreactant role, which is generally used in ECL systems. It was found that only a weak ECL signal (line a in Fig. 2A) was observed on the bare GCE when the potential was cycled between 0 and 1.60 V. However, a strong ECL signal, about 7 times than that of the bare GCE, was obtained at 1.30 V on the PNVs modified GCE (line b in Fig. 2A). These results suggested that the ECL signal originated from the system of PNVs. Furthermore, the ECL signal remained at an almost constant value during consecutive cyclic potential scanning (Fig. 2B), indicating the good reversibility of the modified electrode. Additionally, the ECL working conditions were optimized and shown in Fig. S4. The optimal parameters were used throughout this work. The ECL spectra were also measured by employing a series of optical filters, and a distinguished ECL spectrum peak at approximately 627 nm was observed (Fig. 2C). It was different with that
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Fig. 2. (A) ECL responses obtained on a bare (a) and PNVs modified (b) GCEs. (B) Time-dependent ECL signals of the PNVs modified GCE. (C) ECL spectrum of PNVs. All ECL signals were collected in 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl.
obtained on the bare GCE in the same condition (Fig. S5), suggesting that the PNVs played a key role in the ECL mechanism. In addition, compared to the PL emission spectrum peak of the PNVs at 559 nm, there was a significant spectral shift between the ECL and PL. The red shift was also observed in the previously reported ECL of Si (Ding et al., 2002) and CdSe NCs (Myung et al., 2003), which was explained to the strong role of surface states on the ECL process of the NCs. This indicated that the prepared PNVs had a sensitive surface state. In order to explore the ECL mechanism of the PNVs, the effect of various conditions on the ECL behavior of PNVs modified GCEs was further investigated. When K2S2O8 was absent in reaction solutions, no appreciable ECL signal was observed (Fig. S6), indicating that S2O82 played an important role in the ECL process as a coreactant. Moreover, the PNVs modified GCE was also scanned between þ2.0 and 1.6 V. The anodic ECL response was not found, and the cathodic ECL signal was basically consistent with that obtained between 0 and 1.6 V (Fig. S7). This suggested the coreactant ECL contributed to the whole ECL dominantly. In addition, the ECL response was not obviously changed in oxygen-depleted conditions (not shown), revealing that the ECL buildup was not primarily on the presence of oxygen. All of the above ECL properties indicated that the PNVs ECL was coreactant mechanism. In all coreactant ECL system toward the light emission, four processes generally are involved, namely (a) redox reactions at
electrode, (b) homogenous chemical reactions, (c) excited state species formation and (d) light emission (Miao, 2008). Herein, when the potential is swept to a sufficiently negative potential, an electron from the working electrode might be injected to PNVs to generate the PNVsd (Fig. S8). Meanwhile, the coreactant S2O82 could also be reduced during this cathodic process to SO4d and SO42 on the PNVs modified GCE (Fig. S9). The formed intermediate SO4d would subsequently react with PNVsd to produce the excited state PNVs* via electron transfer from PNVsd to SO4d . Another possibility is that the strongly oxidizing species SO4d might oxidize PNVs to PNVsd þ , which is analogous to the formation of aromatic hydrocarbons radical cation upon oxidation by SO4d (Fabrizio et al., 2000). Subsequently, the annihilation of PNVsd þ and PNVsd ions generates the excited sate PNVs* and PNVs. Finally, PNVs* returns to the ground state accompanied with photon irradiation, thus the ECL emission could be obtained. The possible ECL processes of the PNVs are described with the following equations: PNVsþ e -PNVsd
(1)
S2 O82 − + e− → SO42 − + SO4⋅−
(2)
PNVs⋅− + SO4⋅− → PNVs⁎ + SO42 −
(3)
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Fig. 3. (A) ECL profiles of the PNVs modified GCE in the presence of different DA concentrations in 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl. (B) Linear calibration plot for DA detection.
and/or
PNVs + SO4⋅− → PNVs+ + SO42 −
(4)
PNVs þ þPNVsd -PNVs* þPNVs
(5)
finally, PNVsn-PNVs þhν
(6)
3.3. Application of ECL from PNVs To demonstrate the sensing performance of the PNVs modified GCE, dopamine (DA, an important neurotransmitter) was chosen as a model analyte. When a trace of DA was injected into the 0.2 M PBS (pH 7.2) containing 0.1 M K2S2O8 and 0.1 M KCl, the ECL emission showed an increase (Fig. 3A). Because the surface of the PNVs consist of aromatic stacking arrangements (Yan et al., 2010), so DA is easy to be adsorbed on the PNVs due to strong π–π interaction. The adsorption of DA on the PNVs surface could accelerate the electron transfer (Wang, Y., et al., 2009) from the DA directly to the PNVs, which finally might lead to the enhancement of ECL signal (Yuan et al., 2014). The improved current response could be obtained at the PNVs modified electrode in the presence of DA (Fig. S10), indicating that DA could indeed facilitate the electrochemical behavior in this system. With increasing the concentration of DA, the ECL intensity increased linearly from 10 to 200 pM (n ¼10, R¼0.9998). When further increasing the concentration of DA, the ECL response reached to a saturated value. The corresponding calibration curve (Fig. 3B) could be well fitted with the following equation:
I /I0 = 1.0003 + 0.0098 × CDA (pM)
(7)
where I0 is the initial ECL intensity, I is the ECL intensity at a given concentration of DA. The detection limit was estimated to be 3.15 pM for DA at a signal/noise ratio of 3, which is much lower than those of some inorganic NCs such as CdSe (0.5 mM) (Liu et al., 2008) and Ag2Se (0.1 mM) (Cui et al., 2012), as well as Au (∼1.0 mM) (Hu and Xu, 2010) and Ag (0.92 nM) (Liu et al., 2013) nanoclusters. These results indicate that the prepared PNVs modified GCE could be used for the monitoring of trace amounts of DA.
An interference investigation was also performed by using the solution containing 100 pM DA and 200 pM common co-existing substances such as uric acid, ascorbic acid, glucose, sucrose, urea, cysteine, catechol, acetone, Na þ and Cu2 þ , respectively. No significant change (less than 71%) was observed. Furthermore, increasing the concentrations of interference reagents to be 0.5 mM, the interferences were less than 73%. The good selectivity for DA might be attributed to the efficient electron transfer between DA and PNVs (Li et al., 2011, Wang, G.L., et al., 2009). In addition, five PNVs modified GCEs prepared under the same conditions showed an acceptable reproducibility with a relative standard deviation (RSD) of 3.14% for the detection of 100 pM DA. The prepared PNVs modified GCE maintained 96.1% of the original response after storage in dark and 4 °C refrigerator for 4 weeks, indicating the good stability of the sensor. In addition, the practical drug (dopamine hydrochloride injection) was also measured based on the PNVs modified electrode. After diluting the injection solution, the concentration of DA in the solution was detected to be 19.89 70.25 pM by ECL, close to the given dilute solution value of 20 pM. The relative standard deviation (RSD) for five measurements was 1.43%. These results indicate that this method has acceptable selectivity and accuracy.
4. Conclusions In conclusion, ECL was first observed from the PNVs derived from dipeptide building blocks. The ECL mechanism of the PNVs was possibly ascribed to cathodic coreactant mechanism. The highly sensitive detection for the model analyte DA has been obtained based on increasing ECL intensity of the PNVs modified GCE. Although the ECL intensity of the PNVs modified GCE was not very high at present, preliminary experimental results are promising, indicating that PNVs could be a new class of material for ECL sensing. More importantly, the present study opens up a new avenue to design and development of ECL devices from bioinspired materials. The variety of amino acids and formed diverse nanostructures would provide the endless combinatorial options for these biomaterials. Finally, we believe that the use of the bioinspired nanomaterials with the inherent biocompatibility and capability of molecular recognition would bring huge potential in the ECL bioanalytical applications.
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Acknowledgments This work was supported by the National Basic Research Program of China (No. 2011CBA00508) and the National Natural Science Foundation of China (No. 21175009).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.07.060.
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