Multichannel Electrochemiluminescence of Luminol in Neutral and

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Anal. Chem. 2004, 76, 4002-4010

Multichannel Electrochemiluminescence of Luminol in Neutral and Alkaline Aqueous Solutions on a Gold Nanoparticle Self-Assembled Electrode Hua Cui,* Yang Xu, and Zhi-Feng Zhang

Department of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China

The electrochemiluminescence (ECL) behavior of luminol on a gold nanoparticle self-assembled electrode in neutral and alkaline pH conditions was studied under conventional cyclic voltammetry (CV). The gold nanoparticle selfassembled electrode exhibitied excellent electrocatalytic property and redox reactivity to the luminol ECL system. In neutral solution, four ECL peaks were observed at 0.69, 1.03, -0.45, and -1.22 V (vs SCE) on the curve of ECL intensity versus potential. Compared with a bulk gold electrode, two anodic and one cathodic ECL peaks were greatly enhanced, and one new cathodic ECL peak appeared. In alkaline solution, two anodic ECL peaks were obtained at 0.69 and 1.03 V, which were much stronger than those on a bulk gold electrode. These ECL peaks were found to depend on gold nanoparticles on the surface of the electrode, potential scan direction and range, the presence of O2 or N2, the pH and concentration of luminol solution, NaBr concentration, and scan rate. The emitter of all ECL peaks was identified as 3-aminophthalate by analyzing the ECL spectra. The spatial distribution of the luminol ECL peaks on the gold nanoparticle self-assembled electrode was studied by CCD. The surface state of the gold nanoparticle self-assembled electrode was characterized by scanning electron microscopy (SEM) and UV-visible reflection spectra. The mechanism for the formation of these ECL peaks has been proposed. The results indicate that the gold nanoparticle self-assembled electrode could lead to novel ECL properties, and strong luminol ECL in neutral and alkaline solutions could be obtained on such an electrode, which is of great analytical potential. Electrogenerated chemiluminescence (ECL) has become an important and valuable detection method in analytical chemistry in recent years.1 ECL of luminol, lucigenin, tris(2,2′-bipyridyl) ruthenium (II) and their analogues on various conventional electrodes, such as glassy carbon, paraffin-impregnated graphite, gold, and platinum electrodes, has been documented.2-12 At such * To whom correspondence should be addressed. E-mail: [email protected]. (1) Fa¨hnrich, K. A.; Pravda, M.; Guibault, G. G. Talanta 2001, 54, 531-559. (2) Cui, H.; Zou, G. Z.; Lin, X. Q. Anal. Chem. 2003, 75, 324-331. (3) Sun, Y. G.; Cui, H.; Lin, X. Q. Chin. J. Anal. Chem. 1999, 27, 497-503. (4) Cui, H.; Zhang, Z. F.; Zou, G. Z.; Lin, X. Q. J. Electroanal. Chem., in press. (5) Li, F.; Lin, X. Q.; Cui, H. J. Electroanal. Chem. 2002, 534, 91-98. (6) Li, F.; Cui, H.; Lin, X. Q. Luminescence 2002, 17, 117-122.

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conventional electrodes, ECL-based detection suffers from several problems. For example, basic pH value is usually required for a luminol ECL system in order to achieve high ECL intensity, and the light emission is very weak in neutral solution.1 Therefore, it is difficult to use luminol ECL for the detection of biologically important compounds occurring at physiological pH. Moreover, the electrode surface is readily contaminated, resulting in poor analytical reproducibility. Previous work has revealed that the behavior of ECL is strongly affected by a number of factors, including the applied potential, electrode material, and surface state of the electrode.2-12 Therefore, it is expected that ECL-based detection can be improved through optimization of the composition and surface structure of the electrode. Nanoparticle self-assembled electrodes have received considerable attention in electrochemistry and electroanalytical chemistry due to their fascinating electrochemical, electrocatalytic, redoxreactive, molecule-identifying, and biocompatible properties.13-15 Although Bard’s research group has reported electrogenerated chemiluminescence from octanol-, octane-, and octanethiol-capped Si and trioctylphosphine oxide (TOPO)-capped CdSe nanocrystals in organic solvents, the study concerning ECL on nanoparticle self-assembled electrodes has not been reported until now. It seems that nanoparticle self-assembled electrodes are inherently ideal for ECL because of an enormous surface area-to-volume ratio, which is highly susceptible to heterogeneous redox chemistry with surrounding environments.16,17 The unique properties of nanoparticle self-assembled electrodes that are different from conventional electrodes may lead to new ECL properties and new ECL reactions. In this work, we assembled gold nanoparticles on a bulk Au electrode via cysteine and explored the ECL behaviors of luminol on a gold nanoparticle self-assembled electrode. It was found that (7) Haapakka, K.; Kankare, J. J. Anal. Chim. Acta 1982, 138, 263-275. (8) Haapakka, K. Anal. Chim. Acta 1982, 141, 263-268. (9) Zu, Y. B.; Bard, A. J. Anal.Chem. 2001, 73, 3960-3964. (10) Xu, X. H.; Bard, A. J. Langmuir 1994, 10, 2409-2414. (11) Cui, H.; Wu, L. S.; Chen, J.; Lin, X. Q. J. Electroanal. Chem. 2001, 504, 195-200. (12) Ikkai, H.; Nakagama, T.; Yamada, M.; Hobo, T. Bull. Chem. Soc. Jpn. 1989, 62, 1660-1662. (13) Kastner, M. A. Phys. Today 1993, 46, 24-31. (14) Lewis, L. N. Chem. Rev. 1993, 93, 2693-2730. (15) Brown, K. R.; Fox, A. P.; Natan, M. J. J. Am. Chem. Soc. 1996, 118, 11541157. (16) Ding, Z. F.; Quinn, B. M.; Haram, S. K.; Pell, L. E.; Korgel, B. A.; Bard, A. J. Science 2002, 296, 1293-1297. (17) Myung, N.; Ding, Z. F.; Bard, A. J. Nano Lett. 2002, 2, 1315-1319. 10.1021/ac049889i CCC: $27.50

© 2004 American Chemical Society Published on Web 06/23/2004

luminol in neutral and alkaline aqueous solutions exhibited outstanding ECL properties under cyclic voltammetric (CV) conditions. In neutral aqueous solution, the intensity of ECL peaks at the anode was greatly enhanced, and a strong new ECL peak appeared at the cathode. In alkaline aqueous solution, two anodic ECL peaks much stronger than those on a bulk gold electrode were observed. The spatial distribution of ECL peaks was studied by CCD images. The spectra of various ECL peaks at different potentials were analyzed. The surface status of the gold nanoparticle self-assembled electrode was characterized with SEM and UV-visible reflection spectra. The effects of various factors, such as potential scan direction and range, N2, O2, pH, and NaBr concentration, on luminol ECL peaks were examined. Finally, a possible mechanism for luminol ECL peaks on the gold nanoparticle self-assembled electrode has been proposed. EXPERIMENTAL SECTION Chemicals and Solutions. Luminol was obtained from Merck (Germany). A 1.0 × 10-2 mol/L stock solution of luminol was prepared by dissolving luminol in 0.1 mol/L sodium hydroxide solution without purification. Working solutions of luminol were prepared by diluting the stock solution. HAuCl4‚4H2O (48% w/w) was obtained from Shanghai Reagent (Shanghai, China). A 1.0 g/L HAuCl4 stock solution was prepared by dissolving 1 g HAuCl4 in 1 L of redistilled water and stored at 4 °C. Phosphate buffer solutions (PBS, 0.1 mol/L) with various pH values were prepared by mixing stock standard solutions of K2HPO4 and KH2PO4 and adjusting the pH with 0.1 mol/L H3PO4 or NaOH. Nitrogen and oxygen of 99.999% purity were used. All other reagents were of analytical grade, and redistilled water was used throughout. Synthesis of 16-nm Au Particles. Gold colloids with a diameter of 16 nm were prepared according to ref 18. A 50-mL portion of HAuCl4 (10-2 % w/w) solution was heated to boiling. While stirring vigorously, 1 mL of trisodium citrate (1 wt %) was added rapidly. The solution was maintained at the boiling point for 15 min, during which time a color change from gray to blue to purple was observed before a wine-red color was reached. The heating source was removed, and the colloid was kept at room temperature for 15 min and then stored at 4 °C. The resulting gold nanoparticles were characterized by transmission electron microscopy (TEM) (Hitachi H-800, Japan) and UV-visible spectra (Shimadzu UV-2401 PC spectrophotometer, Japan). Statistical analysis of TEM data revealed that the average diameter of the gold colloids was ∼16 ( 1 nm. The UV-visible spectra exhibited a well-developed surface plasmon absorption peak at 510 nm, with an extinction coefficient () of 2.30 × 108 cm-1 mol/L. Fabrication of Gold Nanoparticle Self-Assembled Electrode. Cysteine has been used to immobilize gold nanoparticles on a platinum microelectrode.19 To our knowledge, a mercaptogroup reacts readily with Au to form the stable Au-S bond. Accordingly, we assembled gold nanoparticles on a bulk Au electrode via cysteine. The electrode was polished with abrasive paper, rinsed with ethanol and redistilled water, and then dried with filter paper. The cleaned electrode was first immersed in 0.1 mol/L cysteine aqueous solution for 2 h at room temperature in darkness. The resulting monolayer-modified electrode was rinsed (18) Frens, G. Nat. Phys. Sci. 1973, 241, 20-22. (19) Li, H.; Wen, J. X.; Cai, Q.; Wang, X. L.; Xu, J. M.; Jin, L. T. Analyst 2001, 126, 1747-1750.

thoroughly with redistilled water and soaked in redistilled water for 12 h to remove the physically adsorbed cysteine, then it was dipped into the colloidal gold for 24 h at 4 °C. Finally, the gold nanoparticle self-assembled electrode was dipped into redistilled water for conservation at 4 °C. The surface state of the gold nanoparticle self-assembled electrode was characterized by SEM (JEOL JSM-6700F, Japan) and UV-visible reflection spectra (Shimadzu UV-365 spectrophotometer, Japan). Electrochemical and ECL Measurements. ECL and electrochemical (EC) measurements were performed by a homemade ECL/EC system, including a model CHI832 electrochemical working station (Chenhua Inc., Shanghai; i.e., the Chinese distributor of CH Instruments Inc., Austin, TX), an H-type electrochemical cell (homemade), a model 1P21 photomultiplier tube (PMT) (Beijing, China), a model GD-1 luminometer (Xi’an, China), and a computer. The H-type ECL cell was constructed as described previously.2 A gold foil with 6.8 × 7.0 mm2 modified with 16-nm gold nanoparticles served as the working electrode, a platinum wire as the counter electrode, and a silver wire as the quasireference electrode (AgQRE). A Ag quasireference electrode (AgQRE) was used due to simplicity for cell construction and quick potential response. Although the potential of the AgQRE was found to be essentially stable during an experiment, measurements of ∆E ) EAg/Ag+ - ESCE in different solutions were taken for potential calibrations. To the working compartment and the auxiliary compartment of the ECL cell, a 2.0-mL portion of the sample solution and the blank solution without luminol were filled in, respectively. When the potential was applied to the working electrode, an ECL signal was generated. The curves of ECL intensity versus applied potential (IECL/E) and the curves of current versus applied potential (i/E) were recorded simultaneously. Under a nitrogen or an oxygen atmosphere, nitrogen or oxygen were bubbled through the solutions for 15 min in both compartments of the cell, and the flow was maintained over the solution during experiments. All experiments were carried out at ambient room temperature. ECL Spectra Measurements. CL spectra of various ECL peaks at different potentials were measured by inserting filters at wavelengths of 360, 380, 400, 420, 430, 470, 490, 510, 535, 550, 565, 580, 600, 630, 650, 670, and 700 nm (light cannot pass at wavelengths lower than these values) under CV conditions. ∆Ifλ was calculated as shown: e.g., ∆If360 ) Iblank(without filter) - I360, ∆If380 ) I360 - I380, ∆If400 ) I380 - I400, etc. The curves of ∆Ifλ versus λ are consistent with CL spectra.2,20 CCD Measurement. CCD photos were taken from a Roper Scientific CCD (Princeton Instruments, Trenton, NJ). The exposure time was 15 s, and the CCD camera was cooled to -32.5 °C. Moreover, a CCD array allowed us to take a full 2D picture without scanning. The beam was finally focused on a CCD matrix of 512 × 512 photodetector. The type of camera selected was EEV 576 × 384 (ph) with focus of 1 m. RESULTS AND DISCUSSION Electrochemical and ECL of Luminol under Air-Saturated Conditions. Cyclic voltammograms (CVs), semidifferential CVs, (20) Lin, J. M.; Yamada, M. Anal. Chem. 1999, 71, 1760-1766.

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Figure 2. Effect of electrolytes on CV (A) and IECL-E (B) curves of luminol. Electrolytes: 0.1 mol/L NaNO3 (- - -), 0.1 mol/L NaBr (- ‚‚ -), 0.1 mol/L NaCl (- - -), 1×10-3 mol/L NaI (-). Inset shows the enlarged CVs from 0.90 to -0.10 V. Figure 1. (A) CV curves on a bulk gold electrode in blank solutions (‚‚‚) and in sample solution (s) and on a gold nanoparticle selfassembled electrode in blank solution (- - -) and in sample solution (-‚‚-) under an air-saturated atmosphere. (B) IECL/E curves of luminol on a bulk gold electrode (s) and on a gold nanoparticle selfassembled electrode (-‚‚-) under an air-saturated atmosphere. NaBr, 0.1 mol/L; pH ) 7.0 in PBS; luminol, 1×10-4 mol/L. Scan rate, 40 mV/s. Inset (a) shows the enlarged semidifferential voltammograms from 0.60 to 0.75 V, inset (b) shows the enlarged CVs from 0.72 to -0.51 V, and inset (c) shows the enlarged ECL-1 and ECL-2 on a bulk gold electrode. If not mentioned additionally, all high voltages applied to the PMT were maintained at -550 V.

and IECL/E curves of luminol in neutral solution containing NaBr on a gold nanoparticle self-assembled electrode and on a bulk Au electrode under air-saturated conditions are shown in Figure 1. In CVs and semidifferential CVs (Figure 1A), two anodic cyclic voltammetric peaks (cvp1, cvp2) were observed at 0.67 and 1.15 V (vs SCE), respectively, on the positive scan. Upon reversal of the potential scan from 1.52 V, four cathodic peaks (cvp3, cvp4, cvp5, cvp6) were found at 0.66, 0.47, -0.45, and -0.95 V, respectively. Among them, cvp1 disappeared in blank solution without luminol (inset (a) in Figure 1A), corresponding to the oxidation of luminol. Cvp2 and cvp3 that could also be observed on a bulk Au electrode were attributed to the oxidation and reduction of nanogold because they appeared in blank solution without luminol.6 Cvp2 was probably also involved in the oxidation of Br-.2 However, it was difficult to distinguish the oxidation peak of Br- from that of nanogold under this condition. When NaI displaced NaBr as an electrolyte, the oxidation peak of I- appeared at a more negative potential separated from that of nanogold, which showed indirect evidence for the oxidation of Br- (Figure 2A). Cvp4 was supposed to be the reduction of BrO- because the 4004 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

peak disappeared when NaNO3 was used as an electrolyte instead (Figure 2A). Cvp5 was the reduction of the dissolved oxygen in solution to HOO- according to our previous studies on luminol ECL at a glassy carbon electrode.3 Cvp6 was the reduction of PO43-, which was observed in PBS but not in boracic acid buffer solution. Finally, the onset of H2 was at about -1.49 V. In IECL/E curves on a gold nanoparticle self-assembled electrode (Figure 1B), corresponding to two anodic voltammetric peaks (cvp1 and cvp2), ECL-1 and ECL-2 were observed at 0.69 and 1.03 V, respectively, on the positive scan. On the reverse scan, corresponding to cathodic voltammetric peak cvp5 and the potential of H2 onset, ECL-3 and ECL-4 were obtained at -0.45 and -1.22 V, respectively. It must be highlighted that ECL-1 and ECL-2 on a bare Au electrode were very weak and were enhanced by ∼2 to 3 orders of magnitude on a gold nanoparticle selfassembled electrode, as shown in Figure 1B, which demonstrated that nanogold could catalyze ECL-1 and ECL-2. Moreover, ECL-4 could only be observed on a gold nanoparticle self-assembled electrode, implying that nanogold was necessary for the generation of ECL-4. CCD Image. A spatial distribution of electrochemiluminescence on a gold nanoparticle self-assembled electrode was studied by CCD. Figure 3 is a collection of ECL images of a gold nanoparticle self-assembled electrode during one CV scan, corresponding to four ECL peaks shown in Figure 1. The luminescent dots seem to be discrete on the surface of the gold nanoparticle self-assembled electrode. We suppose that the luminescent dots occur on gold nanoparticles, which might behave as an ensemble of closely spaced but isolated microelectrodes.15,21 Moreover, it (21) Cheng, W. L.; Dong, S. J.; Wang, E. K. Langmuir 2002, 18, 9947-9952.

Figure 3. CCD image of luminol ECL on a gold nanoparticle self-assembled electrode during one CV scan. Exposure time: 15 s. Recording time: (A) 5 s, corresponding to ECL-1; (B) 25 s, corresponding to ECL-2; (C) 80 s, corresponding to ECL-3; (D) 105 s, corresponding to ECL-4.

Figure 4. SEM images of a bare Au electrode (A) and a gold nanoparticle self-assembled electrode (B).

can be observed that the light emission of ECL-1, ECL-2, and ECL-3 was restricted on the surface of the electrode; however, ECL-4 was significantly diffused around the electrode. It indicates that the electrogenerated species might be stirred vigorously by the released H2 bubble from the surface of the electrode at such high negative potentials. Surface State of Gold Nanoparticle Self-Assembled Electrode. The surface state of a gold nanoparticle self-assembled electrode was characterized by SEM and UV-visible reflection spectra. Figure 4 demonstrates the SEM images of a gold nanoparticle self-assembled electrode and a bulk Au electrode. The nanometer-sized gold particles or clusters were distributed evenly and formed almost a continuous monolayer on the surface of the gold nanoparticle self-assembled electrode. Statistical analysis of SEM data demonstrated that the average diameter of the gold nanoparticles was ∼22 ( 6 nm, which was larger than the average diameter, 16 nm, of the gold colloids. Additionally, during the procedure of focusing, gold nanoparticles were damaged by the beam of electrons, which led to the faint images of Au nanoparticles22 and the error in calculating the average diameter of the gold nanoparticles. This could be one reason that the average diameter, 22 nm accounted according to Figure 4B, is somewhat larger than the gold colloid size. UV-visible reflection spectra of gold nanoparticles self-assembled on an Au electrode and the unmodified Au surface were quantitatively compared, and the difference spectrum obtained after subtracting UV-visible reflection spectrum of the unmodified Au surface is shown in Figure 5. The results show that there is a surface plasmon absorption peak at 460 nm, which has a 50-nm blue shift (22) Grabar, K. C.; Brown, K. R.; Keating, C. D.; Stranick, S. J.; Tang, S. L.; Natan, M. J. Anal. Chem. 1997, 69, 471-477.

Figure 5. Difference spectrum obtained by subtracting UV-visible reflection spectrum of the unmodified Au surface from UV-visible reflection spectrum of gold nanoparticle self-assembled on a Au electrode.

compared with that of Au colloid in aqueous solution. This blue shift may be due to the interaction between the gold nanoparticles and matrix.23 These results indicate directly the formation of a gold nanoparticle self-assembled electrode. ECL Stability and Reproducibility on Gold Nanoparticle Self-Assembled Electrode. The stability of this gold nanoparticle self-assembled electrode was examined, as shown in Figure 6. Experimental results indicate that the gold nanoparticle selfassembled electrode is very stable for the response of both electrochemistry and ECL, even if the cyclic voltammetry was performed via scanning in the wide range of 0.12 f 1.52 f 0.12 f -1.49 f 0.12 V (vs SCE). The validity of a gold nanoparticle self-assembled electrode could reach as long as 30 days if it is stored in redistilled water. The repulsive electrostatic forces kept the Au nanoparticles on the surface of the electrode from aggregation.24 Moreover, for bulk Au, Pt, glassy carbon, and paraffin-impregnated graphite electrodes, the contamination of the electrode surface is a serious problem, and the pretreatment of the electrode surface must be carefully carried out before each measurement so that reproducible ECL can be obtained. However, excellent reproducibility of ECL can be achieved on the gold nanoparticle self-assembled electrode without any pretreatment, and thus, tedious pretreatment can be avoided. ECL Spectra of Various ECL Peaks. The ECL spectra of peaks 1-4 in the IECL/E curves were analyzed under air-saturated (23) Link, S.; El-Sayed, M. A. Rev. Phys. Chem. 2000, 19, 409-453. (24) Hu, X. Y.; Xiao, Y.; Chen, H. Y. J. Electroanal. Chem. 1999, 466, 26-30.

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Figure 8. CV (A) and IECL/E (B) curves of luminol under air-saturated conditions with an initial cathodic scan direction. Luminol, 1×10-4 mol/L; NaBr, 0.1 mol/L; pH ) 7.0 in PBS. Scan rate, 40 mV/s.

Figure 6. CV (A) and IECL/E (B) curves on a gold nanoparticle selfassembled electrode freshly prepared (s) and stored for 7 days (-‚‚-) and 30 days (‚‚‚).

Figure 7. ECL spectra of ECL peaks at different potentials. Conditions as for Figure 1. ECL-1, 0.69 V (A); ECL-2, 1.03 V (B); ECL-3, -0.45 V (C); ECL-4, -1.34 V (D).

conditions, as shown in Figure 7. The results show that the maximum emission of all peaks is ∼425 nm, corresponding to the light emission of 3-aminophthalate.1 It is concluded that all ECL peaks are initiated by luminol reactions. Effect of Various Factors. Various factors influencing luminol ECL, such as potential scan direction and range, the presence of N2 or O2, the pH and concentration of luminol, the concentration of NaBr, and the scan rate, were studied systematically. When the potential scan direction was changed, i.e., from 0.12 f -1.49 f 0.12 f 1.52 f 0.12V (Figure 8), ECL-1 and ECL-2 appeared, and both intensityies increased slightly. However, ECL-3 and ECL-4 disappeared. The results reveal that ECL-1 and ECL-2 4006 Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

Figure 9. CV (A) and IECL/E (B) curves of luminol under air-saturated (‚‚‚), O2 (-‚‚-), and N2 (s) atmospheres. Luminol, 1 × 10-4 mol/L; NaBr, 0.1 mol/L; pH ) 7.0 in PBS. Scan rate, 40 mV/s.

might be enhanced by species produced on the negative potential scan, whereas the compound produced during the positive potential scan could be necessary for ECL-3 and ECL-4. The effect of the potential scan range on luminol ECL was studied. The switching potential was selected as ∼0.96, ∼1.17, and ∼1.52 V. If the reversal of the potential scan was from 0.96 V (after cvp1), then ECL-2, ECL-3, and ECL-4 were invisible. The

Figure 10. CV (A) and IECL/E (B) curves of luminol on a bulk Au electrode in pH 13.0 NaOH solutions (s) and on a gold nanoparticle selfassembled electrode in pH 11.0 (- - -) and 13.0 (-‚‚-) NaOH solutions. CV (D) and IECL/E (C) curves of luminol on a bulk Au electrode in pH 13.0 NaOH solutions (s) and on a gold nanoparticle self-assembled electrode in pH 7.4 PBS (-‚-‚). Inset (a) shows the enlarged ECL on a bulk Au electrode in pH 13.0 NaOH solutions, and inset (b) shows the enlarged ECL on a gold nanoparticle self-assembled electrode in pH 7.4 PBS. NaBr, 0.1 mol/L; luminol, 1×10-4 mol/L. Scan rate, 40 mV/s. High voltage applied to the PMT, -280 V.

results suggest that electrochemical or chemical reactions after cvp1 initiate ECL-2, ECL-3, and ECL-4 reaction. ECL-3 and ECL-4 were obtained from the scan switched from ∼1.17 V. Moreover, the intensities of ECL-3 and ECL-4 were enhanced with an increase in the scan range. Therefore, it is deduced that the oxidation products of Br- are essential to ECL-3 and ECL-4, because when we displaced NaBr with NaNO3, ECL-3 and ECL-4 almost disappeared (Figure 2B). Earlier studies showed that Br- was readily oxidized to BrO- at higher positive potential.25 Therefore, BrOmight be formed in the potential range of ∼0.96 to 1.17 V, and it was difficult to distinguish the oxidation peak of Br- from that of nanogold. In an oxygen atmosphere, ECL-1 and ECL-2 increased slightly, whereas ECL-3 and ECL-4 increased significantly. In a nitrogen atmosphere, ECL-1 and ECL-2 decreased remarkably, whereas ECL-3 and ECL-4 almost disappeared (Figure 9B). The results suggest that O2 could significantly enhance ECL-1 and ECL-2 and is necessary for the formation of ECL-3 and ECL-4. ECL-3 is supposed to correlate to OOH- formed by the reduction of O2 at the negative potentials. Therefore, it is easier to understand the effect of O2 on ECL-3. The effect of the pH of luminol solution on IECL/E and CV curves of luminol was examined in the pH range of ∼5.8 to 13.0. Typical CV and IECL-E curves of luminol are shown in Figure 10 (25) Peng, G. Z.; Wang, G. Z. Handbook for Electroanalytical Chemistry (Chinese); Chemical Engineering Press (China): Beijing, 1999; pp 162-163.

(a high voltage as low as -280 V was applied to the PMT in order to compare ECL intensities with each other under different pH conditions, and thus, ECL intensity in physiological pH was weak) and others in supporting materials. It seems that all ECL peaks were related to the pH of the luminol solution. When the pH was lower than 5.8, ECL-1, ECL-2, ECL-3, and ECL-4 were almost invisible. The intensity of all ECL peaks increased with an increase in pH (Figure 10B). The intensity of ECL-1 and ECL-2 reached a maximum at pH 11.0, whereas ECL-4 increased significantly in the pH range of ∼6.0 to 8.0 and then changed slightly beyond pH 8.0. The maximal emission occurring at pH 11.0 on a gold nanoparticle self-assembled electrode was much stronger than that occurring at pH 13.0 on a bulk gold electrode. For example, ECL-1 on a gold nanoparticle self-assembled electrode was enhanced by ∼2 orders of magnitude in alkaline solutions. Although the emission in physiological pH 7.4 on a gold nanoparticle selfassembled electrode was weaker than maximal emission at pH 13.0 on a bulk gold electrode (Figure 10C), the emission intensity in neutral solution on a gold nanoparticle self-assembled electrode was still good enough, because only a high voltage of -280 V was applied to the PMT (maximum of high voltage is -900 V). The number of ECL peaks also changed with the pH of the solution. At pH ∼6.0 to 9.2 (PBS buffer), four ECL peaks were observed. At pH ∼10.0 to 12.0 (NaOH medium), only two anodic ECL peaks were obtained, and two cathodic ECL peaks disappeared. At pH 13.0 (NaOH medium), ECL-1 was visible, but the Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

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peak potential shifted negatively, whereas ECL-2 became broad emission. Moreover, the reproducibility of ECL became worse at pH 13.0. The results indicate that strong luminol ECL can be obtained on a gold nanoparticle self-assembled electrode under both neutral and alkaline conditions. Luminol concentrations over the range ∼1 × 10-8 to 4 × 10-4 mol/L at pH 7.0 were tested. It was found that the intensity of all ECL peaks increased with an increase in luminol concentration, indicating that all ECL peaks depended on luminol. Moreover, the intensity of ECL-1 as an example was measured, and found to be linear with the concentration of luminol in the range of ∼1×10-8 to 4×10-4 mol/L. Therefore, at pH 7.0, a concentration of luminol as low as 1 × 10-8 mol/L is detectable by use of ECL1. At pH 8.0, the intensity of ECL-1 was studied in the luminol concentration over the range ∼1 × 10-10 to 1 × 10-7 mol/L. The intensity of ECL-1 was also linear with the concentration of luminol, and the detectable concentration of luminol could reach to 1 × 10-10 mol/L. With a continuous increase in pH, it is deduced that the concentration would decrease, because stronger emission was observed. The results demonstrate that luminol ECL still has good emission efficiency in neutral solutions. The effect of NaBr concentrations was also studied. ECL-1 decreased obviously with an increase in NaBr concentration, whereas ECL-2, ECL-3, and ECL-4 increased. Halide ions are wellknown fluorescence quenchers. The quenching of ECL-1 by Brmay follow similar mechanism. When Cl- and I- displaced Br(Figure 2B), the magnitude of the quenching effect increased in the following order: Cl- < Br- < I-, which was in good agreement with the fact that heavier halide ions caused a stronger quenching effect. ECL-2 tended to increase with an increase in NaBr concentration, which may be due to the fact that ECL-2 was related to the oxidation of Br-. At higher positive potentials, Br- was probably oxidized to BrO-,2 followed by the reaction with luminol radical anions to generate light emission. BrO- is likely to be produced in the potential range of ∼0.95 to 1.01 V, because no light emission was found with potential scans reversed from 0.95 V or scans reversed from 1.40 V in solutions without NaBr. The redox products of Br- were also probably involved in the reactions of ECL-3 and ECL-4; thus, a higher concentration of Br- led to an increase in ECL-3 and ECL-4. IECL/E and CV behaviors at scan rates from 20 to 150 mV/s were studied, as well. The peak potential of oxidative peaks such as cvp2 shifted to more positive potentials with an increase in scan rate, whereas the peak potential of reductive peaks, such as cvp4 and cvp6, shifted to more negative potentials. Compared with the ECL intensity at different scan rates, ECL-1 and ECL-2 increased obviously with an increase in scan rate, whereas ECL-3 and ECL-4 changed irregularly. Mechanism of Luminol ECL at Gold Nanoparticles SelfAssembled Electrode. Luminol ECL at the Anode. ECL-1, corresponding to cvp1, was caused by the electrooxidization of luminol anions to luminol radicals. The experimental results showed that higher pH values, luminol concentrations, and scan rates were favorable for the occurrence of ECL-1. ECL-1 could be enhanced by oxidative species such as O2 and O2•- and decreased with an increase in halide ion concentration. The mechanism for ECL-1 is likely to be similar to ECL-1 that occurred at a bulk Au electrode in an alkaline solution.2 Unlike bulk gold, 4008

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gold nanoparticles exhibited remarkable catalysis in the ECL-1 process. In the presence of O2, the pathways could be

ECL-1: LH- - e- f LH• f L•- + H+ (Ep ) +0.67 V)

(1)

L•- + O2 f O2•- + L

(2)

L•- + O2•- f LO22-

(3)

LO22- f AP2-* + N2

(4)

AP2-* f AP2- + hν

(5)

From eq 1, higher pH and luminol concentration favor the formation of luminol radicals. That higher Br- concentrations lead to a decrease in ECL-1 is probably due to the fact that excitedstate AP2-* is transferred into the excited triplet state.2 If the initial scan direction was negative, OOH- was produced at negative potentials.26

O2 + H2O + 2e- f OOH- + OH- (Ep ) -0.45 V) (6) Upon the reverse scan, OOH- was oxidized to O2•- (eq 7), which could enhance ECL-1 via eqs 3, 4, and 5.

OOH- - e- f HOO• / O2•-

(7)

ECL-2 was related to Br- because ECL-2 could not be found when NaBr was displaced with NaNO3. It seems that ECL-2 was induced by BrO- because Br- existed in the solution initially, but light emission related to Br- was only observed at positive potentials higher than 1.05 V, implying the oxidation of Br-. The experimental results show that higher pH values and higher Brconcentrations were favorable for the occurrence of ECL-2, but the N2 atmosphere was not beneficial for ECL-2. Accordingly, ECL-2 caused by the reaction of luminol radicals with BrO- may follow the pathways

ECL-2: Br- + 2OH- - 2e- f BrO- + H2O (Ep ) +1.05 V)

(8)

2L•- + 3BrO- + 2OH- f f 2AP2-* + 3Br- + H2O + N2 (9) From eq 8, higher pH and luminol concentration are advantageous to the formation of ECL-2; whereas in the presence of N2, the balance of eq 9 shifted toward the left-hand side, resulting in a decrease in ECL-2. Gold nanoparticles can strongly catalyze ECL-1 and ECL-2. ECL reactions are considered to be a combination of electrode reactions and subsequent chemical reactions. Therefore, there were two possibilities for the catalytic mechanism. First, the electrode reaction is catalyzed by nanogold via accelerating direct (26) Vitt, J. E.; Johnson, D. C.; Engstrom, R. C. J. Electrochem. Soc. 1991, 138, 1637-1643.

electron transfer (ET) between an electrode and a redox reactant. Gold nanoparticles are highly susceptible to heterogeneous redox chemistry with the surrounding environment because of their enormous surface-area-to-volume ratios.16 For example, Natan and co-workers showed that direct, reversible cyclic voltammetry of horse heart cytochrome c (Cc) in solution was obtained at uncoated submonolayers of 12-nm-diameter colloidal Au particles on SnO2, and the colloidal particles behave as an ensemble of closely spaced but isolated microelectrodes, whereas the electrochemical oxidation of Cc was slow at a conventional electrode.15 Second, subsequent chemical reaction is catalyzed. Corain reviewed that gold nanoparticles could catalyze the reactions in aqueous solution.27 In this case, the high current intensity of cvp1 (inset (a) in Figure 1A) observed for luminol on a gold nanoparticle self-assembled electrode was indicative of facile electron transfer, and thus, strong ECL-1 was due to electrocatalytic activity of the gold nanoparticle self-assembled electrode. However, the electrode process corresponding to ECL-2 was invisible in CVs. Therefore, for ECL-2, between the electrode reaction and the subsequent CL reaction, which step is catalyzed by nanogold is difficult to determine at this moment. Luminol ECL at the Cathode. ECL-3 depended on the dissolved oxygen and corresponded to cvp5 at -0.45 V. It is believed that cvp5 is the reduction of the dissolved oxygen in solution to OOH-.26 Therefore, ECL-3 is likely to be due to the reaction of luminol with OOH-. The pathways for ECL-3 could be

ECL-3: O2 + H2O + 2e- f OOH- + OH- (Ep ) -0.45 V) (10) LH- + OOH- f f AP2-*

(11)

ECL-3 was also related to Br-, because when NaNO3 was used for the experiments instead of NaBr, ECL-3 significantly decreased. If the initial scan direction started positively, BrO- was produced at a higher positive potential. Previous studies have demonstrated that luminol could react with electrogenerated HO2and ClO- to undergo stronger light emission.2 Likewise, we believed that ECL-3 was enhanced by the following reaction:

LH- + OOH- + BrO- f f AP2-* + Br- + H2O + N2 (12) This is supported by the experimental fact that the higher the Br- concentration, the higher the ECL-3 intensity; ECL-3 was very weak in the absence of Br-. Moreover, earlier studies indicated that oxygen dissociation over nanogold was more difficult, and thus, the formation of surface OOH- should be intensified,28 which might explain the fact that ECL-3 was enhanced on a gold nanoparticle self-assembled electrode. The mechanism of ECL-4 is somewhat complicated. O2 and Br- were crucial to initiate ECL-4 because ECL-4 was not generated under a N2 atmosphere or in the absence of Br-. Moreover, ECL-4 was not found on a bare Au electrode, which suggested that nanogold was essential to the formation of ECL-4. (27) Schmid, G.; Corain, B. Eur. J. Inorg. Chem. 2003, 17, 3081-3098. (28) Haruta, M. Catal. Today 1997, 36, 153-166.

With a negative initial potential scan, ECL-4 was not observed. Therefore, it is deduced that ECL-4 correlated to the redox product of Br- during a positive potential scan and corresponding reverse scan. If NaI was used instead of NaBr, when starch was added to the solution, the color of the solution changed to blue at about -0.20 V, confirming that I2 was formed. Accordingly, there might be Br2 electrogenerated at high negative potentials. At more negative potentials, the dissolved oxygen was reduced to HO2-. On the other hand, ECL-4 was generated at the potential of H2 onset. If ECL-4 only relates to Br2 and HO2-, it should occur at less negative potentials. It seems that H (g) formed at such high negative potentials also took part in ECL-4 reactions. The probable mechanism for ECL-4 is as follows.

ECL-4: BrO- + H2O + e- f Br2 + 2OH-

(13)

H+ + e- f H (g)

(14)

LH- + Br2 + H (g) + nanogold + HO2- f AP2-* + H- + X + Y (15)

X and Y represent the redox product of Br2 and the other product, respectively. At present, the overall reaction scheme for ECL-4 is not clear because most of the reactants in ECL-4 are intermediates electrogenerated at different potentials; as a result, it is extremely difficult to identify them. Further work is under way. CONCLUSIONS Four luminol ECL peaks in the pH range of ∼6.0 to 9.2 and two ECL peaks in alkaline solutions were found on a gold nanoparticle self-assembled electrode under conventional CV conditions. The mechanism for these ECL peaks has been proposed due to the reactions of luminol radical electrooxidized by luminol or luminol with the dissolved oxygen and various electrogenerated species, such as O2•-, OOH-, BrO-, Br2, and H (g) at different potentials. Compared with a bulk Au electrode, the gold nanoparticle self-assembled electrode can remarkably catalyze luminol ECL at the anode in both neutral and alkaline solutions and initiate one new cathodic ECL peak in the pH range of ∼6.0 to 9.2. Moreover, it can offer excellent stability and reproducibility and avoid tedious pretreatment of electrode surface. Present work shows that the gold nanoparticle selfassembled electrode is unique in its catalytic property and electrochemical reactivity with respect to the luminol ECL reactions. Strong luminol ECL is for the first time obtained in neutral pH, implying that luminol ECL on the gold nanoparticle selfassembled electrode is of great potential for the sensitive detection of biologically important compounds occurring in physiological pH due to the excellent biocompatible property of nanogold. ACKNOWLEDGMENT The support of this research by the National Natural Science Foundation of P.R. China (Grant Nos. 29875025 and 20375037) and the Overseas Outstanding Young Scientist Program of Chinese Academy of Sciences are gratefully acknowledged. We Analytical Chemistry, Vol. 76, No. 14, July 15, 2004

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are also grateful to Prof. S. Gao and Mr. J. J. Ding for taking the CCD images.

luminol, and at different scan rates. This material is available free of charge via the Internet at http://pubs.acs.org.

SUPPORTING INFORMATION AVAILABLE

Received for review January 18, 2004. Accepted March 31, 2004.

IECL/E curves and CV curves of luminol in different buffer solutions, at different pH values, at different concentrations of

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