In Situ Activation of CdS Electrochemiluminescence Film and Its

Aug 6, 2014 - An electrochemiluminescence sensor based on a sulfur-terminal CdS 2 L complex. Chunyan Zhang , Lei Wang , Shengyi Zhang , Changjie ...
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In Situ Activation of CdS Electrochemiluminescence Film and Its Application in H2S Detection Yan-Yan Zhang, Hong Zhou, Peng Wu, Huai-Rong Zhang, Jing-Juan Xu,* and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, China S Supporting Information *

ABSTRACT: Nanocrystals (NCs) usually suffer from weak electrogenerated chemiluminescence (ECL) emissions compared with conventional luminescent reagents like Ru(bpy)32+. In this work, we proposed a simple in situ activation approach by dipping CdS NCs film on glass carbon electrode (CdS NCs/GCE) in an activation solution containing H2O2 and citric acid, resulting in a ∼58-fold enhancement of ECL intensity in the presence of coreactant H2O2. During activation, CdS NCs were oxidized by H2O2 to smaller ones which resulted in more surface S vacancies; meanwhile, citric acid played an important role in stabilizing NCs. The ECL enhancing mechanism was investigated in detail, and the coordination of H2O2 to surface excess Cd2+ ions (S vacancies) on the CdS NCs surface formed in activation was the main factor which could stabilize the electrogenerated radicals, resulting in an enhanced ECL. ECL from the activated CdS NCs/GCE could be quenched in Na2S solution due to the bonding of S(II) to excess Cd2+ ions on the surface of CdS NCs. On the basis of this, we then used the activated CdS NCs/GCE as an ECL probe for the detection of Na2S which showed good performance including a wide linear range of 5 nM to 20 μM and good anti-interference ability. Moreover, this ECL probe was successfully applied for hydrogen sulfide (H2S) detection in a biological system.

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Incorporating nitrogen in TiO2 nanotubes (NTs) could also greatly enhance the ECL intensity by 10.6-fold and make the ECL spectrum red shift which was due to valence band edge shifting upward.10,11 Moreover, several nanomaterials such as multiwalled carbon nanotubes6 and K-doped graphene12 promoting the electron transfer between NCs and the electrode have been applied in improving the ECL behaviors of NCs by 5.3- and 2.3-fold, respectively. Additionally, due to their excellent conductivity, Au NPs13 and Ag NPs14 can also accelerate the electron-injecting rate in an ECL reaction and ECL emissions were enhanced by 17- and 5-fold, respectively. Since ECL is so sensitive to surface states, it is possible that ECL could also be improved by controlling the surface chemistry through chemical treatment.15 In this work, we first achieved greatly enhanced ECL emissions by in situ activation of the CdS NCs film in the presence of H2O2 and citric acid. Compared with the unactivated CdS NCs thin film, ca. 58 times enhanced ECL intensity was achieved for the activated CdS NCs thin film modified on glass carbon electrode (GCE) in the presence of coreactant H2O2, which was much greater than those obtained from methods discussed above. The enhancement was due to more surface S vacancies and the

lectrogenerated chemiluminescence (ECL), which is generated by an electrochemical reaction between electrogenerated species, has emerged as a powerful analysis tool because of its simplified optical setup, low background signal, excellent temporal and spatial controllability, and no need of a light source.1,2 Since the first report on the ECL study of silicon nanoparticles (NPs) by Bard et al.,3 semiconductor nanocrystals (NCs) have been exploited as a new kind of ECL emitter. Compared with conventional molecular emitters, NCs have several distinctive merits such as size/surface-trap controlled luminescence and good stability against photobleaching.4 However, NCs usually suffer from relatively weaker ECL emissions than those of conventional luminescent reagents like luminol or Ru(bpy)32+ which restrict their wide analytical applications. Thus, finding a way to obtain high ECL efficiency of NCs for bioanalysis is the constant driving force of this area. Many previous works have demonstrated that ECL emissions of NCs mainly occur via surface electron−hole recombination and are highly dependent on their surface states which can be changed by doping metal ions or composition with other nanomaterials.5,6 Generally, incorporating metal ion dopants in semiconductors can enhance the luminescence efficiency by creating a new electron energy level or perturbing the host energy level.7 It was reported that the doping of Mn2+ or Eu3+ ions in the CdS NCs surface could alter the surface of CdS NCs and create a new surface state-Mn2+ or Eu3+ complex,5,8,9 producing a maximum 4-fold enhancement in ECL intensity. © XXXX American Chemical Society

Received: April 26, 2014 Accepted: August 6, 2014

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Scheme 1. Schematic Representation of Activation of CdS NCs by H2O2 and Citric Acid (a) and Its Application in H2S Detection Based on the Quenching Effect of ECL Emissions Caused by Adsorption of H2S on the Surface S Vacancies (b)

coordination of H2O2 to surface excess Cd2+ ions when detected in the presence of the coreactant H2O2 which stabilized electrogenerated coreactant-related radical OH•, resulting more excited states (CdS)*. Moreover, after activation, NCs film became thinner and less dense than before which exposed more surface S vacancies and made it possible for ECL to occur not only at the interface but also in the interior of the NCs film. H2S, known as a toxic gas with its unpleasant smell of rotten eggs, exists in our body and other biological systems in small amounts and connects importantly with biological functions. It has been recognized as the third gaseous signaling molecule besides nitric oxide (NO) and carbon monoxide (CO).16,17 Deregulation of H2S has been linked to many diseases, such as Down syndrome,18 Alzheimer’s diseases,19 diabetes,20 and liver cirrhosis.21 As a result of the huge potential benefits of understanding the biological functions of H2S, the research of H2S has received considerable attention. Accurate and reliable measurements of H2S concentration in biological samples are needed and can provide useful information to understand its functions. A number of methods have already been developed, such as colorimetric,22 electrochemical,23 gas chromatography,24 fluorescent,25 and phosphorescent26 assays. However, there is still a pressing need to develop new simple techniques for the rapid, sensitive, and selective detection of H2S in biological media. Here, we found that ECL emissions from the activated CdS NCs/GCE sharply decreased after being incubated in Na2S solution which was due to the bonding of S(II) to excess Cd2+ ions on the surface of CdS NCs as shown in Scheme 1. Depending on this ECL signal decrease, the activated CdS NCs/GCE was then used as an ECL probe for the detection of Na2S which showed a wide linear range of 5 nM to 20 μM and good anti-interference ability. Moreover, this ECL probe was successfully applied for H2S detection in biological media.

solution of Na2S·9H2O (0.5960 g) in 30 mL of ultrapure water was slowly injected to form a mixture of which the molar ratio of Cd2+ to S2− was 1:4, and instantly orange-yellow precipitates were obtained. The reaction was held at 70 °C for 3 h with continuous refluxing. The final reaction precipitates were centrifuged and washed thoroughly with absolute ethanol two times and ultrapure water two times. Then, the obtained precipitate was ultrasonically dispersed into ultrapure water for centrifugation at 9000 rpm for 10 min to collect the upper yellow colloidal solution of CdS NCs. The final dispersion with a concentration of 2.3 mg/mL was stored at 4 °C for further use. Preparation and Activation of CdS NCs Modified GCE. GCE was first polished with successively finer grades of SiC papers and then with 0.3 μm of alumina powder to obtain a mirror-like surface. Then, GCE was thoroughly rinsed with water and sonicated in ultrapure water. By dropping 10 μL of the CdS NCs dispersion onto the pretreated surface of GCE and evaporated in air at room temperature, we fabricated the CdS NCs/GCE which was stored in 0.1 M PBS buffer (pH 7.4) for further use. To activate the surface of CdS NCs, the CdS NCs/GCE was immersed in 0.5 mL of activation buffer containing 5.0 mM H2O2 and 4.85 × 10−2 M citric acid (pH 5.0 adjusted by 0.2 M Na2HPO4) under gentle vibration for 16 h at 37 °C. After activation, the activated CdS NCs/GCE was washed with ultrapure water and used for ECL detection. ECL Sensing for Hydrogen Sulfide. The activated CdS NCs/GCE used as an ECL probe was first immersed in 0.5 mL of 0.1 M PBS buffer (pH 7.4) containing various concentrations of Na2S under gentle vibration at 37 °C for 7 min and washed with 0.1 M PBS buffer (7.4) to remove nonspecifically adsorbed ions. Then, ECL measurement was conducted in 0.1 M PBS buffer (pH 8.5) containing 5.0 mM H2O2. Detection of Hydrogen Sulfide Concentration in Calf Serum. According to the literature,25 with slight modification, 1 μL of different concentrations of Na2S (0, 0.45, 0.9, 1.8 mM) was spiked into 90 μL of new born calf serum, respectively. Then, 60 μL of CH3CN was added respectively to precipitate the protein in serum, and the samples were centrifuged at 6000 rpm for 1 min. After that, 140 μL of supernatant liquid was added into 280 μL of CH3CN/H2O (40% CH3CN) to precipitate proteins again and then the solution was centrifuged

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EXPERIMENTAL SECTION Reagents and Apparatus. Refer to the Supporting Information. Synthesis of CdS NCs. CdS NCs were prepared according to the literature with a slight modification.5 In brief, Cd(NO3)2· 4H2O (0.1861 g) was dissolved in 30 mL of ultrapure water and heated to 70 °C under stirring. Then, a freshly prepared B

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Figure 1. (A) The ECL-potential curves of the original CdS NCs/GCE (a), CdS NCs/GCE after being treated in activation buffer containing 5.0 mM H2O2 and 4.85 × 10−2 M citric acid (b), CdS NCs/GCE after being treated in 4.85 × 10−2 M citric acid (pH 5.0 adjusted by 0.2 M Na2HPO4) (c), CdS NCs/GCE after being treated in 5.0 mM H2O2 (d) at 37 °C for 16 h, and the activated CdS NCs/GCE after being incubated in 20 μM S2− solution at 37 °C for 7 min (e). ECL detection buffer: 0.1 M PBS buffer (pH 8.5) containing 5.0 mM H2O2. Inset: amplified plot (a) and (c). (B) Corresponding cyclic voltammograms (CVs) of (a), (b), and (e) detected in 0.1 M PBS buffer (pH 8.5) containing 5.0 mM H2O2. Inset: CVs of (a) and (b) detected in 0.1 M PBS buffer (pH 8.3) containing 0.05 M K2S2O8.

at 6000 rpm for 5 min. Ultimately, 100 μL of supernatant liquid sample was added into 400 μL of ultrapure water, and the activated CdS NCs/GCE was incubated in it at 37 °C for 7 min. After being washed, the ECL signal of the activated CdS NCs/GCE was obtained in 0.1 M PBS buffer (pH 8.5) containing 5.0 mM H2O2.

ment of the ECL efficiency of CdS NCs in an H2O2 system for bioanalysis is greatly needed. In this work, we are excited to have found that, if the CdS NCs/GCE was immersed in the activation buffer containing both H2O2 and citric acid, the activated CdS NCs/GCE exhibited outstanding ECL properties, the ECL intensity of which was ca. 58 times higher than that of the original CdS NCs/GCE detected in 0.1 M PBS buffer containing 5.0 mM H2O2 (Figure 1A, curves a and b). However, it was worth noting that, if the original CdS NCs/GCE was soaked in the buffer containing only citric acid or H2O2, the ECL emission was enhanced only 76.3% (Figure 1A, curve c) or 3.6 times (Figure 1A, curve d) compared to that of the original CdS NCs, respectively. These results suggest that H2O2 together with citric acid played an important role in enhancing the ECL intensity of CdS NCs. To make the activation effect more quantitative, ECL quantum efficiency or yield, φECL, defined here as photons emitted per electron passed,32 is estimated. Due to the complexity of coreactant involved ECL systems, the irreversible nature of coreactant electrochemistry, and the high concentrations of coreactants in solution compared to ECL emitters, the absolute value of φECL is hardly measurable.1 Thus, relative ECL efficiencies of CdS NCs modified on GCE before and after activation were calculated by using a standard 0.05 mM Ru(bpy)32+/5.0 mM TPrA system and the relationship φECL = φECL ° (IQ°/I°Q), where φECL ° is the ECL efficiency of the standard system, I and I° are the integrated ECL intensities of CdS NCs film/5.0 mM H2O2 and 0.05 mM Ru(bpy)32+/5.0 mM TPrA systems, and Q and Q° are the charges passed (in coulombs) for the above two systems, respectively.33,34 The ECL-potential curve and corresponding CV of the 0.05 mM Ru(bpy)32+/5.0 mM TPrA system were shown in Figure S2, Supporting Information, and 100% is assigned to the value of φ°ECL. Relative ECL efficiencies, φECL of 0.78% for the original CdS NCs film/5.0 mM H2O2 system and 55.1% for the activated CdS NCs film/5.0 mM H2O2 system, were obtained, respectively. Thus, after activation, the relative ECL efficiency of CdS NCs film in the 5.0 mM H2O2 system was enhanced ca. 70 times. From eq 3, we can see that the ECL intensity was highly dependent on the probability of collision between reduced NC (CdS)•− and coreactant related radical OH• which was higher if

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RESULTS AND DISCUSSION ECL Enhancing Mechanism. CdS NCs are one of the most popular ECL emitters of II−VI semiconductor NCs due to their intrinsic properties such as easy preparation in aqueous solution and potential applications in analytical fields.27,28 H2O2 is an important coreactant for semiconductor NCs-based cathodic ECL emissions. In the presence of coreactant H2O2, as the electrode potential became sufficiently negative, the CdS NC was reduced to (CdS)•−, and the coreactant H2O2 was reduced to OH• which could react with (CdS)•− to obtain an excited state (CdS)*. This state emitted light in the aqueous solution to produce an ECL signal.8,29 The formation of intermediate OH• in the H2O2 evolved ECL process was verified by EPR spectra (Figure S1, Supporting Information). Thus, the ECL processes can be described by the following equations: CdS + ne− → n(CdS)•−

(1)

H 2O2 + e− → OH− + OH•−

(2)

(CdS)•− + OH• → (CdS)* + OH−

(3)

(CdS)* → CdS + hv

(4)

As the ubiquitous dissolved oxygen can be electrochemically reduced to H2O2, it can act as a ready-made coreactant for ECL emissions without the introduction of exogenous coreactants which makes the system neat, green, and facile.4 Besides, as O2 and H2O2 both are the substrates of oxidases and peroxidases, NCs-based ECL biosensors can be fabricated on the basis of enzyme-catalytic reactions.12,30 However, H2O2 has weaker oxidizing capacity and exhibits a lower efficiency than S2O82−.31 Compared with conventional luminescent reagents like Ru(bpy)32+, CdS NCs usually exhibit relatively weak ECL emissions especially in the presence of H2O2 as a coreactant which may restrict their analytical applications. Thus, enhanceC

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H2O2. However, when detected in the presence of K2S2O8 as a coreactant, after activation, the cathodic peak potential in CV of CdS NCs/GCE shifted slightly (from −1.19 to −1.22 V) (inset in Figure 1B) and the ECL emission intensity or relative ECL efficiency was only ca. 7 times or 13 times higher than that of the original CdS NCs/GCE (Figures 2 and S4B, Supporting Information), which further demonstrated the significance of adsorption of the coreactant H2O2 or O2. Figure 3A showed the ECL spectra of the original CdS NCs and the activated CdS NCs. As can be seen, there were two distinctive emission peaks appearing on the ECL spectra which can be interpreted in Figure 3B according to the literature.35 One peak appearing at around 500 nm belonged to the annihilation of electron in the surface trap with the hole in the valence band; the other one at 620 nm was also observed, which can be attributed to annihilation of electron in the surface trap and surface hole trap. The position of each peak hardly changed, but the ECL intensity at every detected wavelength was greatly increased, which contributed to the total ECL enhancement. The activation of CdS NCs was further tested with PL spectroscopy. As shown in Figure S5, Supporting Information, a broad emission was observed for both the original and activated CdS NCs in the range of wavelengths from 450 to 650 nm with a peak at around 510 nm. Compared to the absorption band edge position of the original CdS NCs dispersion at 471 nm (Figure S6, curve a, Supporting Information), the broad spectrum and distinct red shift of PL emission indicated that the PL was mainly due to the surface trap emission.36,37 Surface defects, which generally originate from surface S2− dangling bonds, could lead to a nonradiative recombination of the excitons and PL quenching.38 After activation, the PL peak intensity was obviously enhanced, demonstrating the reduced number of S2− dangling bonds through the oxidation by H2O2. Because of the small size of CdS NCs we synthesized, the change in particle size of NCs film modified on GCE after activation could not be finely characterized from scanning electron microscope (SEM) images. Thus, UV−vis absorption spectra and photographs of the original and treated CdS NCs dispersion were tested. After being mixed with treating buffer containing only citric acid (pH 5.0), CdS NCs were coagulated which was rather different from the original homogeneous dispersion (inset A in Figure S6, tubes a and b, Supporting Information) and the absorption edge position shifted from 471 to 481 nm (Figure S6, curves a and b, Supporting Information). According to the Peng’s empirical equation,39 the diameters of original CdS NCs (a) and citric acid treated CdS NCs (b) were estimated to be 6.23 and 6.71 nm. The coagulation was caused by the instability of CdS NCs in acidic solution. If CdS NCs were treated in H2O2 solution, from inset A in Figure S6, tube c, Supporting Information, it was obvious that most of them became really small and the diameter of them was estimated to be 4.69 nm as a result of the oxidation by H2O2 according to the absorption edge position at 436 nm (Figure S6, curve c and inset B, Supporting Information). If CdS NCs were treated in the activation buffer containing both H2O2 and citric acid, the color of the activated CdS NCs dispersion was faded (inset A in Figure S6, tube d, Supporting Information). The absorption edge position of the activated CdS NCs (d) underwent a blue shift to 448 nm (Figure S6, curve d, Supporting Information), and the diameters of them were estimated to be 5.19 nm. The decrease in the size of CdS NCs and relatively homogeneous solution after activation indicates the synergistic effect of H2O2

the radicals were more stable. Therefore, ECL emission performance of CdS NCs was closely related to the stability of the reduced NCs. The electrochemical behaviors of CdS NCs film before and after activation in deaerated 0.1 M PBS buffer were shown in Figure S3, Supporting Information. It was clearly observed that after activation the anodic peak current became distinct, suggesting that the reduced CdS NCs formed in the negative direction scan were stable enough to undergo oxidation on scan reversal. Due to such stability of the reduced CdS NCs, more (CdS)* was formed and thus a more intense luminescence was obtained. In addition, according to the literature, the oxidation of the surface of CdS NCs by H2O2 could cause the formation of more surface S vacancies.35 To demonstrate this, the activated CdS NCs/GCE was incubated in 20 μM S2− solution at 37 °C for 7 min and a sharp decrease (56.4%) of ECL emissions was observed as shown in Figure 1A, curve e. The great ECL decrease was due to S(II) being able to be bonded to excess Cd2+ ions on the surface of CdS NCs. Besides, according to the report,35 H2O2 can be adsorbed on surface S vacancies when the activated CdS NCs/GCE was soaked in the ECL detection buffer containing coreactant H2O2. From the corresponding CVs (Figure 1B), we can see that after activation the reduction peak potential of H2O2 shifted negatively (from −0.90 V (curve a) to −1.17 V (curve b)) and this tendency could be inhibited after being incubated in the S2− solution (curve e), really indicating that H2O2 was strongly adsorbed on the S vacancies of the NCs surface during the ECL detection process. The bonding action between the surface of Cd2+ and H2O2 stabilized the electrogenerated coreactant-related radical OH•, making them not easy to annihilate.8 Thus, more (CdS)•− could be oxidized to excited states by OH• and resulted in improved ECL efficiency. This explanation could be further confirmed by replacing coreactant H2O2 by O2 or K2S2O8. As shown in Figures 2 and S4A, Supporting Information, when detected in air saturated 0.1 M PBS buffer (pH 8.5) (O2 as a coreactant), the ECL signal intensity or relative ECL efficiency of the activated CdS NCs was ca. 52 times or 61 times higher than that of the original CdS NCs, which corresponded with the ECL increase when detected in PBS buffer containing

Figure 2. Effects of different kinds of coreactants (H2O2, O2, and K2S2O8) on the ECL enhancement efficiency ΔI/I0 = (I − I0)/I0 or Δφ/φ0 = (φ−φ0)/φ0. (I0 and I are the ECL intensities from the original and activated CdS NCs modified on GCE, respectively; φ0 and φ are the relative ECL efficiencies of the original and activated CdS NCs modified on GCE using the 0.05 mM Ru(bpy)32+/5.0 mM TPrA system as a standard, respectively.) D

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Figure 3. (A) ECL spectrum of the original CdS NCs (a) and the activated CdS NCs (b) obtained through filters under a cyclic potential scan from 0 to −1.2 V. ECL detection buffer: 0.1 M PBS buffer (pH 8.5) containing 5.0 mM H2O2. (B) Interpretation of two ECL emission peaks of CdS NCs modified on GCE.

Figure 4. 3-dimensional AFM images and thickness of unmodified (A and B), original CdS NCs film modified (C and D), and the activated CdS NCs film modified (E and F) silicon wafer.

and citric acid. On one hand, the oxidation by H2O2 caused the particle size to decrease and abundant surface S vacancies remained on the surface of NCs. The inconspicuous extinction absorption peak can be attributed to the surface traps on the CdS NCs.37 On the other hand, Cd2+ ions on the surface of NCs could be coordinated by citric acid which stabilized CdS NCs.40 It was worth noting that the size of the activated CdS NCs (d) was not as small as that of the H2O2 treated CdS NCs (c), strongly suggesting that such coordination on the surface of NCs by citric acid protected NCs from being too greatly oxidized by H2O2. TEM images of the original CdS NCs and the activated CdS NCs were shown in Figure S7, Supporting Information. According to the TEM observation, the average size of CdS NCs was decreased from 6.59 ± 0.69 to 5.23 ± 0.62 nm, which was consistent with the absorption spectra. To reveal the morphology of the CdS NCs film before and after activation, the AFM technique was then employed using a bare silicon wafer. The silicon wafer was smooth and flat as shown in Figure 4A,B. When the CdS NCs film was modified on the wafer, the thickness increased to ca. 210 nm but the surface remained smooth like a piece of cake (Figure 4C,D). However, after being activated in the presence of H2O2 and citric acid, the thickness of the NCs film decreased to ca. 52 nm while the roughness was enhanced greatly (Figure 4E,F). The significant differences in the morphology demonstrated that after activation the NCs film became much thinner and less dense than before which not only provided more surface area and exposed more surface S vacancies but also facilitated the

diffusion of H2O2 into the membrane and resulted in greatly enhanced ECL. Influencing Factors on the ECL Enhancement Efficiency. In order to further investigate the mechanism of activation, we then studied the effects of H2O2 concentration, citric acid amount, pH, and other small organic acids on the ECL enhancement efficiency ΔI/I0 of CdS NCs. Figure 5A shows the relationship between H2O2 concentration and ECL enhancement efficiency of CdS NCs. It was found that, the more H2O2 the activation solution contained, the greater was the ECL enhancement we obtained, demonstrating that more S vacancies were formed in the surface of CdS NCs. However, when the H2O2 concentration was as high as 6.0 mM, the CdS NCs film modified on GCE was unstable and dropped into the ECL detection solution ultimately which made it unlikely to be used for further application. Thus, in this work, 5.0 mM H2O2 was adopted for the activation buffer. As shown in Figure 5B, when the concentrations of H2O2 and pH were fixed, as citric acid amount increased, the ECL enhancement efficiency increased greatly which demonstrated the role that citric acid played. Citric acid has one central and two terminal carboxylate groups as well as one central hydroxyl group as illustrated in Table S1, Supporting Information. According to the literature, both the central hydroxyl and carboxylate oxygen atoms can bind to Cd2+ ions, forming a fivemembered ring structure; meanwhile, the central alcoholic oxygen atom together with the terminal carboxylate oxygen atom also coordinates with Cd(II), forming a six-membered E

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Figure 5. Effects of H2O2 (A) and citric acid (B) concentrations and pH (C) on ECL enhancement efficiency ΔI/I0 with (A) the concentration of citric acid fixed at 4.85 × 10−2 M and pH fixed at 5.0, (B) the concentration of H2O2 fixed at 5.0 mM and pH fixed at 5.0, and (C) the concentration of H2O2 and citric acid fixed at 5.0 mM and 4.85 × 10−2 M, respectively. (D) Effects of different kinds of acids on ECL enhancement efficiency ΔI/ I0: (a) citric acid; (b) acetic acid; (c) oxalic acid; (d) succinic acid; (e) DL-malic acid. The concentrations of different acids and H2O2 were fixed at 4.85 × 10−2 M and 5 mM, respectively. ECL detection buffer: 0.1 M PBS buffer (pH 8.5) containing 5.0 mM H2O2.

citric acid and the five- or six-membered ring coordination structure could also be formed; therefore, the ECL enhancement efficiency in the presence of malic acid at pH 5.0 and 5.5 was as substantial as that in the presence of citric acid (Figure 5D, curve e). On the basis of the above results, small organic acids that have relatively low pKa values and the potential of forming stable coordination structures with Cd2+ ions, together with H2O2, are effective for causing a significant enhancement of the ECL intensity of NCs. Under the optimized conditions, the original CdS NCs/GCE was activated and the ECL signal-time curve under continuous potential scanning was shown in Figure S8, Supporting Information. The as-prepared CdS NCs film generated strong and stable ECL emissions in the presence of coreactant H2O2 which was mainly attributed to the highly stable reduced form of CdS NCs.5 Such strong and stable ECL signals made the activated CdS NCs/GCE an excellent platform to construct ECL-based biosensors. Sensing to H2S in Serum. As shown above, ECL emissions from the activated CdS NCs/GCE sharply decreased after being incubated in Na2S solution. This was because of the replacement of citrate ligands by S(II) since the stability constant K of CdS (1.25 × 1026) is much higher than that between Cd2+ and citrate molecules (2 × 1011). This interesting phenomenon may be applied to the detection of H2S. We then used the activated CdS NCs/GCE as a probe and investigated its ECL responses to H2S (using Na2S as the equivalent25) by the ECL quenching efficiency which was defined as −ΔI/I0= −(I − I0)/I0 (%), where I and I0 are the ECL intensities of the activated CdS NCs/GCE in the presence and absence of Na2S, respectively. As shown in Figure S9, Supporting Information, the quenching efficiency −ΔI/I0 (%) reached a platform in 7

ring structure.41,42 Thus, citrate molecules coated on the surface of NCs and played an important role in stabilizing surface S vacancies. However, when the concentration of citric acid increased above 4.85 × 10−2 M, the ECL enhancement efficiency sharply decreased, suggesting that too much coordination could protect the surface of CdS NCs from being further oxidized by H2O2. The role of coordination by citric acid could also be demonstrated by pH effect. As shown in Figure 5C, when the concentrations of H2O2 and citric acid were fixed, the ECL emissions enhanced as the pH increased which was due to more −COOH groups being deprotonated at higher pH and thus causing more coordination structure. Nevertheless, when pH increased to 6.5, the ECL enhancement times decreased quickly. This phenomenon was due to protection as well as the fact that a high pH made H2O2 unstable and it could easily decompose at 37 °C for 16 h. In this work, pH 5.0 was chosen for the activation buffer. To further confirm the mechanism discussed above, we explored the effects on ECL enhancement efficiency of some other small organic acids. As shown in Table S1, Supporting Information, acetic acid has only one −COOH group with relatively high pKa value and thus ECL enhancement efficiency could be ignored (Figure 5D, curve b). As for oxalic acid, it has two adjacent −COOH groups both with low pKa values and thus causing an obvious ECL enhancement efficiency (Figure 5D, curve c). With the carbon chain being longer, the acidity becomes weaker. Thus, succinic acid with two separated −COOH groups and high pKa values also had an insignificant role in improving ECL intensity (Figure 5D, curve d). Due to the inductive effects by −OH on the carbon chain, both pKa1 and pKa2 values of DL-malic acid are comparable to those of F

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Figure 6. (A) ECL signal responses for detection of different concentrations of Na2S: (a) 5 nM, (b) 10 nM, (c) 50 nM, (d) 500 nM, (e) 5 μM, and (f) 20 μM. Inset: linear relationship between ECL quenching efficiency −ΔI/I0 (%) and the logarithm of Na2S concentration, three measurements for each point. (B) ECL quenching efficiency −ΔI/I0 (%) of the activated CdS NCs/GCE in the presence of various interfering agents. (1) 100 μM GSH, (2) 100 μM L-Cys, (3) 100 μM SO42−, (4) 100 μM S2O82−, (5) 100 μM SO32−, (6) 100 μM S2O32−, (7) 100 μM Cl−, (8) 100 μM Br−, (9) 100 μM NO3−, and (10) 5 μM S2−.

surface stabilized the electrogenerated coreactant-related radical OH•, and thus, more (CdS)•− could be oxidized to excited states by OH•, resulting in an improved ECL efficiency. Last but not least, after activation, the NCs film became much thinner and less dense than before. This not only exposed more surface S vacancies as surface area was greatly enhanced but also facilitated the diffusion of small molecules like the coreactant in the ECL detection buffer and made the ECL process occur both at the interface and in the interior of the NCs film. Such activation opened a promising path for other NCs to improve their ECL behaviors and could be further applied to develop highly sensitive ECL sensing systems. On the basis of this in situ activation approach, we developed an ECL sensing system for the sensitive and selective detection of H2S based on the bonding of S(II) to excess Cd2+ ions on the surface of CdS NCs, which has been successfully applied for the detection of H2S concentration in biological media.

min. Thus, we adopted 7 min as the incubation time for the purpose of reproducibility. To demonstrate the efficiency of the probe in determining hydrogen sulfide, varying concentrations of Na2S were tested. As shown in Figure 6A, the ECL intensity of the activated CdS NCs/GCE was very sensitive to the change of Na2S concentration and decreased with the increase in concentration of Na2S, ranging from 5 nM to 20 μM. The ECL quenching efficiency −ΔI/I0 (%) was found to be logarithmically related to the concentration of Na2S in the wide range from 5 nM to 20 μM (shown as the inset in Figure 6A). The regression equation was expressed as −ΔI/I0 = 41.23 + 11.78 log C (C represents the concentration of S2−, μM; correlation coefficient R = 0.991). We next examined the selectivity of the probe by comparing the response to Na2S with the response to common interferences in biological media. It was shown (Figure 6B) that 5 μM Na2S caused an obvious decrease in the ECL intensity of the probe, whereas the existence of a 20-fold excess of other possible interferences, such as glutathione (GSH), Lcysteine (L-Cys), SO42−, S2O82−, SO32−, S2O32−, Cl−, Br−, and NO3−, led to a slight change of the ECL signal. This means that these common interferences did not significantly affect the sensing system to H2S. The results clearly suggested that the activated CdS NCs/GCE could be utilized as a sensitive and selective platform for the detection of the target molecule. Finally, we applied the ECL sensing system in the detection of hydrogen sulfide in new born calf serum. We found that the average hydrogen sulfide concentration in new born calf serum is 8.93 ± 1.40 μM as shown in Table S2, Supporting Information, which was consistent with the H2S level of μM in blood serum that was obtained by fluorescent methods.25,43 We then spiked 5, 10, and 20 μM Na2S separately into three original serum samples, and the recoveries were 81.4%, 86.7%, and 105.0%, respectively, which showed an acceptable reliability of this detection technique.

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ASSOCIATED CONTENT

S Supporting Information *

Reagents and apparatus, EPR spectra verifying the existence of OH• in H2O2 involved ECL processes, ECL measurement of the Ru(bpy)32+/TPrA system, CVs of the CdS NCs film, ECL measurements of the CdS NCs film in O2 and K2S2O8 systems, PL spectra, UV−vis absorption spectra, photos and TEM images of CdS NCs dispersion, structures and pKa values of organic acids, stability of the activated CdS NCs film, optimization of the incubation time, and results of the determination in calf serum. This material is free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-25-83597294. E-mail: [email protected]. Notes

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The authors declare no competing financial interest.

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CONCLUSIONS In this work, we provided a simple in situ activation approach to CdS NCs film based on the synergistic effect of H2O2 and citric acid. The ECL enhancement is attributed to three main reasons. First, after activation, the stability of the reduced CdS NCs was enhanced and thus more excited states of CdS NCs could be formed. Second, such treatment caused the formation of more surface S vacancies. The adsorption of H2O2 on the

ACKNOWLEDGMENTS This work was supported by the 973 Program (Grant 2013CB933802) and the National Natural Science Foundation (21327902, 21025522, 21135003, 21121091) of China.

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