A Spectral Shift-Based Electrochemiluminescence Sensor for

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A Spectral Shift-Based Electrochemiluminescence Sensor for Hydrogen Sulphide Cheng Ma, Wanwan Wu, Yujiao Peng, Min-Xuan Wang, Gang Chen, Zixuan Chen, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04229 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 16, 2017

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Analytical Chemistry

A Spectral Shift-Based Electrochemiluminescence Sensor for Hydrogen Sulphide Cheng Ma, Wanwan Wu, Yujiao Peng, Min-Xuan Wang, Gang Chen, Zixuan Chen* and Jun-Jie Zhu*

State Key Laboratory of Analytical Chemistry for Life Science School of Chemistry and Chemical Engineering Nanjing University, Nanjing 210023, P. R. China *To whom correspondence should be addressed: E-mail: [email protected]

E-mail: [email protected]

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ABSTRACT Classic electrochemiluminescence (ECL) assays relying on the change in luminescence intensity face a challenge in the quantitative analysis of complex samples. We report here the design and implementation of a new sensing strategy using the maximum luminescence wavelength (max) shift as the readout to achieve quantitative detection. This approach includes an ECL luminophore (RuSiO2@GO) and a H2S-sensitive inner filter absorber (CouMC). The absorbance of CouMC illustrates a dependence on the H2S concentration, which induces a Δmax of the ECL luminophore. Both experimental and simulated results suggest that the spectral shift of ECL effectively avoids the interference of the total luminescence intensity fluctuations, enabling a highly reliable quantitative analysis. This spectral shift-based ECL assay strategy offers a wide application potential by extending types of ECL luminophores and absorptive chemodosimeters based on inner filter effect.

Keywords: detection • electrochemiluminescence • inner filter effect • hydrogen sulphide • spectral shift


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Electrochemiluminescence (ECL) is a redox-induced luminescence emission where reactive intermediates generated at electrode surfaces undergo an energetic electrontransfer reaction to form excited states and emit light1-3. The combination of electrochemistry and chemiluminescence endows ECL with many distinctive advantages as an alternative luminescence technique4-6. Specifically, compared with photoluminescence, the absence of excitation light in ECL avoids photobleaching, luminescence impurities and spectral overlap of different luminescence reporters. Moreover, ECL allows great control over the position and time of emission, which is advantageous over chemiluminescence and beneficial for selectivity, multi-analyte detection and imaging. Therefore, ECL has become a commercially successful analytical technique and a versatile tool for the understanding of fundamental questions in many fields7-12. Classic ECL assays rely on the change in electrochemically generated luminescence intensity, which is usually collected by a photomultiplier tube (PMT)13-16. However, the intensity-based strategy faces the challenge of false positive or negative errors during the detection that are associated with the instrumental or environmental changes. To offer a more precise measurement in biological applications, ECL ratiometric sensing was performed for probe-target assays (protein, cancer cell, microRNA, etc.) to eliminate most ambiguities by self-calibration of two emission bands17-20. So far, ratiometric ECL methods include dual-potential mode and dual-wavelength mode, but cross-reaction interference from certain oxidative or reductive intermediates strongly restricts the development of ratiometric ECL detection21. In addition, neither dualpotential nor dual-wavelength ratiometric strategies offer dual signals simultaneously, which may result in measurement errors from fluctuations of intensity. The wavelength of maximum emission (max) in the ECL spectrum is an exclusive and inherent characteristic for a given luminophore22-26. Therefore, using max as the readout can effectively avoid the interference of total luminescence intensity fluctuations, enabling a highly reliable quantitative ECL analysis. Here, we constructed and demonstrated a spectral shift-based ECL system for sulphide sensing in vitro. 3 ACS Paragon Plus Environment


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Hydrogen sulphide (H2S), an important molecule involved in human health and diseases27,28, was chosen as the model analyte. The spectral-shift ECL system includes an ECL luminophore and an inner filter absorber. The ECL luminophore was fabricated by conjugating Ru(bpy)32+-doped silica to oxidized graphene (RuSiO2@GO), and the inner filter absorber was a H2S-responsive chemodosimeter (CouMC)29 (Scheme 1). In the absence of H2S, CouMC showed a significant absorbance at 589 nm while RuSiO2@GO showed a strong ECL emission at 619 nm. The overlap of the ECL emission and the absorption of CouMC led a red-shift of ECL spectra due to the inner filter effect (IFE)30,31. In the presence of H2S, the reaction between H2S and the indolenium C-2 atom of CouMC reduced the absorption of CouMC, and the spectral overlap of the absorption of CouMC and the ECL emission of RuSiO2@GO decreased. The suppressed IFE then induced a spectral blue-shift of the ECL emission of RuSiO2@GO. In consideration of the good stability of ECL spectra and the high selectivity of CouMC to H2S, the spectral shift-based ECL sensing strategy illustrates higher accuracy and selectivity than conventional intensity-based ECL methods.

EXPERIMENTAL SECTION Reagents. Unless otherwise stated, all the other chemicals and reagents used in this study were of analytical grade quality. All reagents and solvents were used as received without further purification. 2,3,3-Trimethylindolenine, 3-iodopropyltrimethoxysilane and tetracycline hydrochloride were purchased from J&K Scientific Ltd. (Beijing China).

Diethyl

malonate,

tripropylamine

(TPA)

and

tris(2,2′-

bipyridyl)dichlororuthenium(II) hexahydrate were purchased from Sigma-Aldrich. 4(Diethylamino)salicylaldehyde was purchased from Energy Chemical (Shanghai China). Phosphorus oxychloride was purchased from Xiya Reagent (Chengdu China). CouMC was synthesized as described in the Supporting Information. Glutathione (GSH), cysteine (Cys) and dopamine were purchased from Aladdin Reagent (Shanghai China). Nicotinamide adenine dinucleotide (NAD+) and tetraethyl orthosilicate (TEOS) 4 ACS Paragon Plus Environment

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were purchased from Sinopharm Reagent (Beijing China). Sodium sulfide nonahydrate were purchased from Nanjing Chemical Reagent. S2- equilibrates with HS- and H2S in biological environments (PBS buffer, pH = 7.4). So all experiments in this work were performed by addition of Na2S as a source of sulphide. The concentration of HS- and H2S can be calculated by dissociation constant (Kd). The distribution fraction () of S2-, HS- and H2S in 100 mM PBS (pH = 7.4) were shown in Table S1.

Apparatus. ECL spectra can be obtained by a homemade ECL spectra acquiring system. The setup consisted of a monochromator (Acton SP2300i, PI) equipped with a spectrograph CCD (PIXIS 400BR_excelon, PI) and a grating (grating density: 300 L/mm; blazed wavelength: 500 nm), a CHI 660D electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China), cuvette holder, a light-tight cover. Scanning electron micrographs (SEM) were measured on an S-4800 scanning electron microscope. Transmission electron micrographs (TEM) were measured on Tecnai12 transmission electron microscope, using an accelerating voltage of 120 kV. UV-vis spectra were recorded on a UV-3600 spectrophotometer (Shimadzu, Kyoto, Japan). NMR spectra were recorded on a Bruker DPX 300 MHz spectrometer with internal standard tetramethylsilane (TMS) and solvent signals as internal references at 298 K, and the chemical shifts (δ) were expressed in ppm and J values were given in Hz. Electrospray ionization mass spectra were recorded on LCMS-2020 (SHIMADZU). High-resolution electrospray ionization mass spectra (HR-ESI-MS) were recorded on an LTQ-Orbitrap XL (Thermofisher). The ECL-potential profiles were recorded on ECL analyzer (Xi’an Remex Analytical Instrument Co., Ltd., China) with a threeelectrode system.

Measurement. ECL spectra measurements of RuSiO2@GO/CouMC system were carried out by the homemade setup. ECL spectra were recorded with a three-electrode system, including a working electrode (carbon paper), a Pt counter electrode, and a Ag/AgCl (saturated KCl) reference electrode. The three electrodes were put into a 5 ACS Paragon Plus Environment


cuvette. The electrolyte is 100 mM PBS (pH = 7.4). The ECL spectra were collected by the CCD when cyclic voltammetry were applied. The electrochemical workstation and the spectrograph CCD were synchronously triggered and operated. ECL spectra were collected by circularly scanning the potential ranging from 1.2 V to 1.8 V at 0.1 V/s with an exposure time for 12 s. To obtain normalized ECL spectra, the ECL intensity was divided by the maximum ECL intensity at the spectral peak. ECL intensity measurement of RuSiO2@GO/CouMC system were performed by conventional ECL setup equipped with PMT. ECL intensity were recorded with a three-electrode system, including a working electrode (glassy carbon electrode), a Pt counter electrode, and a calomel (saturated KCl) reference electrode. The three electrodes were put into a customized cuvette. ECL intensity were collected by PMT when cyclic voltammetry were applied by scanning the potential. All spectra data and intensity data were inspected by Grubbs test to reject outliers. Each sample was measured five times (n = 5). The 95% confidence level was used. Preparation of the RuSiO2@GO composite material: (1) RuSiO2 nanoparticles were synthesized as previously described with a little modification32. First, 1.77 mL of Triton X-100 were mixed with 7.5 mL of cyclohexane and 1.8 mL n-hexanol at 25oC. After 30 min stirring, 340 L 0.04 M Ru(bpy)32+ aqueous solution was added into the mixture. After 30 min stirring, 100 L TEOS was added into the mixture. After 5 min stirring, 60 L NH3H2O was added into the mixture to trigger the polymerization reaction. The reaction was allowed to stir for 24 h at 25oC to obtain RuSiO2 nanoparticles. Then 2 mL acetone were added into mixture and the solution were sonicated for 10 min to break the emulsion. The precipitation was washed three times with ethanol and once with distilled water to remove the residual surfactant molecules and extra Ru(bpy)32+. Finally, RuSiO2 was dispersed with ethanol to a final volume of 4 mL. (2) The modification of amino group on RuSiO2 nanoparticles33. 1 mL of the above RuSiO2 nanoparticles suspension (4.4 mg/mL) was diluted with ethanol to 5 mL followed by the addition of 400 L APTES. After being stirred for 8 h at 30oC, the mixture was centrifuged and washed two times with ethanol and two times with distilled water to 6 ACS Paragon Plus Environment

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remove the excess APTES. Then the amino-terminated RuSiO2 nanoparticles were obtained. (3) Preparation of RuSiO2@GO composite materials. GO was synthesized from natural graphite powder according to a modified Hummer’s method34,35. 9.9 mL of GO aqueous solution (22 mg) was mixed with 500 L of EDC aqueous solution (40 mg/mL), 500 L of NHS aqueous solution (40 mg/mL) and 750 L of aminoterminated RuSiO2 nanoparticles aqueous solution (5.7 mg/mL), followed by stirring for 24 h at 30oC. Then the mixture was centrifuged and washed two times with distilled water. Finally, RuSiO2@GO composite materials was dispersed with distilled water to a final volume of 12 mL (1.6 mg/mL).

RESULTS AND DISCUSSION Characterization of RuSiO2@GO and CouMC. TEM and SEM were used to characterize the morphology of RuSiO2@GO (Figure 1A and B). RuSiO2 was attached on the surface of GO by amido bond36. Doping of Ru(bpy)32+ molecules inside the silica nanoparticle protected them from leakage and provided a signal enhancement owing to the high concentration of Ru(bpy)32+ in nanoparticles32,37. GO not only enhanced the ECL intensity by improving the charge transfer rate but also increased the stability of modified materials through π-π stacking interaction between GO and the carbon electrode. RuSiO2@GO showed a strong ECL emission at 619 nm, which is collected by a spectrometer equipped with a spectrograph CCD (Figure 1C and D). CouMC was selected as the absorber, which was prepared by connecting a coumarin and an indolenium block via an ethylene group. The conjugated indole skeleton in CouMC acted as a color-reporting group. HS-, the main stable form of H2S in the physiological condition (Table S1), can be added to the indolenium C-2 atom of CouMC as a nucleophile and lead to an absorbance change29. In the absence of H2S, CouMC showed a significant absorbance at 589 nm (Figure S1). This band decreased gradually upon the titration of H2S, indicating the nucleophilic attack of H2S toward the indolium group in CouMC, and an apparent color change of CouMC from deep blue to pale yellow was observed. In addition, CouMC displayed a rapid response to H2S (Figure S2). Dynamic 7 ACS Paragon Plus Environment


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absorption spectra tracking of CouMC (10 µM) in the presence of H2S (30-200 µM) suggested that the sensing reaction could be completed within 5 min.

Work principle of the spectral shift-based ECL. RuSiO2@GO was synthesized and modified at the electrode by droplet coating. As shown in Figure 2, during successive cyclic voltammetry scans, the ECL intensity of RuSiO2@GO gradually declined, but

max of ECL spectra remained stable. These results demonstrated that using max as the readout can avoid the interference from intensity fluctuations, which were possibly caused by the electrode passivation or undesired side-reactions. The IFE is a source of errors in ECL analysis30,38, but it can be useful for an ECL sensor by converting the analytical absorption signals to ECL signals39. In this work, the ECL emission of RuSiO2@GO passed through a CouMC solution, and was then measured by the spectrometer. In the absence of H2S, CouMC showed a significant absorbance at 589 nm while RuSiO2@GO showed a strong ECL emission at 619 nm (Figure 3A). The overlap of ECL emission spectra (RuSiO2@GO) and absorption spectra (CouMC) quenched the total ECL intensity, and induced a red-shift of ECL spectra via IFE (Figure 3C and Figure S3). To demonstrate that the spectral red-shift was caused by IFE, transmitted spectra of CouMC were measured in the range of 5-50 M (Figure S4). According to the Beer-Lambert law, the transmitted ECL (𝐼𝑡 ) was expressed: 𝐼t = 𝐼0 (𝜆)𝑇(𝐶CouMC , 𝜆)

(1)

where 𝜆 is the wavelength, 𝐶CouMC is the concentration of CouMC, 𝐼0 (𝜆) is the ECL emission intensity of RuSiO2@GO on the electrode surface, 𝐼𝑡 is the ECL emission intensity of RuSiO2@GO passed through the CouMC solution, and 𝑇(𝐶CouMC , 𝜆) is the transmittance of the CouMC solution. According to the equation 1, the simulated 𝐼𝑡 are obtained with the different concentration of CouMC (Figure 3D and Figure S5). Simulated results were consistent with the experimental results, suggesting that the red-shift of ECL spectra was indeed caused by IFE. IFE not only depends on the spectral overlap between the absorbance of CouMC and the ECL emission of RuSiO2@GO, but also depends on the concentration of 8 ACS Paragon Plus Environment

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CouMC and the optical length of CouMC solution. As shown in Figure 3E and F, ECL spectra gradually blue-shifted when either the concentration of CouMC or the optical length decreased, which confirmed the fact that the max of ECL spectra were affected by IFE. The max in ECL spectra was independent of the total ECL intensity. Figure S6 showed that the ECL intensity changed more than 13-fold when we used two different potentials to produce two different 𝐼0 (𝜆) values, the 𝜆𝑚𝑎𝑥 of ECL spectra, however, remained stable. Thus, these results demonstrated that the spectral shift ECL method was regulated by IFE, thereby avoiding the interference from total ECL intensity fluctuations. To demonstrate that equation 1 does not suffer from undesired emissions, we investigate the ECL performance of CouMC. As illustrated in Figure S7, CouMC had negligible ECL emission compared with Ru(bpy)32+ in the TPA system, which demonstrated that the ECL spectral shift was not caused by the ECL of CouMC. In addition, the shape of the ECL spectra of RuSiO2@GO remained stable in the present of H2S (Figure S8). Although CouMC has a fluorescent emission band at 652 nm, such low quantum yield should not affect the result (0.03 with cresyl violet as reference)29. Thus, the contribution of the fluorescence from CouMC is negligible in this system, and the spectral shift of 𝐼𝑡 was only from IFE.

Accuracy and selectivity of the spectral shift-based ECL sensor. In the presence of H2S, the reaction of H2S with the indolenium C-2 atom of CouMC induced a weaker absorption band, and a smaller spectral overlap between the absorption of CouMC and the ECL emission from RuSiO2@GO (Figure 3B). Thus, the suppressed IFE induced a blue-shift of the ECL spectrum and an increase of the ECL intensity (Figure 4A,B and Figure S9). We compared the ECL intensity readout with the ECL spectra readout responding to H2S. As shown in Figure 4C, the ECL intensity readout are unable to distinguish 5 M H2S samples from 10 M H2S samples according to Student’s tdistribution (n = 5). Large standard deviations primarily came from the fluctuations of the ECL intensity. In remarkable contrast, the ECL spectrum readout can easily 9 ACS Paragon Plus Environment


distinguish 5 M H2S samples from 10 M H2S samples because of the high stability of max (with a small standard deviation). These results were proved by both the simulated and experimental ECL spectra. As illustrated in Figure 4D, the intra-assay precision was investigated by assaying the same H2S level for three similar measurements. The similar ECL spectra demonstrated that the ECL spectrum readout possessed a good reproducibility in spite of obvious intensity fluctuations. This experimental results were supported by the simulated results (Figure 4E), which were obtained according to equation 1. Selectivity is another key issue in achieving quantitative measurements. Tetracycline, dopamine, nicotinamide adenine dinucleotide (NAD+), glutathione (GSH), cysteine (Cys), microcystis aeruginosa, urine and blood were selected as coexistent interfering agents to investigate the selectivity of the spectral shift ECL technique in complex samples (Figure 4F). The ECL intensity presented an obvious decline when the solution contained these abovementioned coexistent interfering agents. This was reasonable because tetracycline, dopamine, NAD+, GSH and Cys were redox agents and could lead to electrochemical and redox side-reactions. Detailed quenching mechanisms have been discussed in previous studies40-43. Because microcystis aeruginosa solution, urine and blood have absorbance in visible light region (Figure S10) and biomolecules in these real samples can block the electron transfer, the ECL intensity were dramatically quenched in these real samples. In contrast, negligible variations of max were observed in the ECL spectra with the addition of each interfering agent except for the blood sample, whose absorbance spectrum showed obvious overlap with the ECL spectrum of RuSiO2. Nevertheless, the interference could be eliminated by the dilution of the blood sample. GSH and Cys possessed high pKa value (≥8.5)44,45, thus H2S was a better nucleophile than biological thiols in neutral medium, owing to its lower pKa value (ca. 7.0). Therefore, CouMC had a better selectivity for H2S over biological thiols. These results indicated that the spectral shift ECL approach was able to selectively detect H2S against coexisting interfering agents.


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The titration and detection of H2S in complex sample via the spectral shift-based ECL method. Titration experiments were performed for quantitative detection of H2S. As shown in Figure 5, although a pseudolinear relationship between the H2S concentration and the ECL intensity was obtained in the concentration range from 1 to 30 M, the large deviation was inevitable (R2 = 0.801, n = 5). On the contrary, ECL spectra blue-shifted linearly with the H2S concentration in the range of 1-30 M (R2 = 0.989, n = 5). As shown in Table S2, the 𝜆𝑚𝑎𝑥 readout exhibited a lower relative standard deviation (RSD) than the ECL intensity readout at each concentration of H2S. These results demonstrated that the spectral shift method possessed superior repeatability due to lower RSD and provided a more accurate linear relation. To demonstrate the practical applicability of the spectral shift-based ECL method, the recovery test was performed by adding H2S into serum samples. The accuracy of the spectral-shift ECL approach in H2S detection was examined and compared with results from the intensity-based method. As shown in Table 1, the recovery rate of the serum samples was 47.9% by intensity-based method, attributed to the blocked redox site on the electrode surface induced by nonspecific adsorption of proteins in the serum samples, which quenched the ECL intensity. However, the spectral-shift ECL approach showed a recovery rate of 99.2%, thereby validating the reliability and practicality of this method.

CONCLUSION In summary, we have demonstrated the ability of a spectral shift-based ECL sensing strategy in an accurate assay of sulphide in vitro, where the ECL emission peak shift is used as the readout instead of the classic ECL intensity. By using this strategy, the interference of the intensity fluctuations in continuous cyclic voltammetry scans is avoided, as well as that from coexisting interference agents in complex serum samples. In addition, repeatability and accuracy are significantly improved, as shown by the experimental and simulated results. Our study not only shows a new readout for ECL 11 ACS Paragon Plus Environment


quantitative analysis technique, but also provides a good start for molecular regulated ECL spectra technique, which is beneficial for ECL sensing and light-emitting devices. Still, the spectral shift-based method has potential for application in single-nanoparticle ECL spectral technique for accurately monitoring analytes against the intrinsic heterogeneities in particle size and local conductivities of the electrode surface.

ASSOCIATED CONTENT Supporting Information. Experimental details including synthesis of the CouMC and its characterizations, as well as additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors [email protected] [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We gratefully appreciate financial support from National Natural Science Foundation of China (21427807, 21335004, 21605081) and the Natural Science Foundation of Jiangsu Province (Grants No BK20160638). The authors thank Professor Dechen Jiang in Nanjing University for the revise of manuscript. REFERENCES (1) Richter, M. M. Chem. Rev. 2004, 104, 3003-3036. (2) Hercules, D. M. Science 1964, 145, 808-809. (3) Ritchie, E. L.; Pastore, P.; Wightman, R. M. J. Am. Chem. Soc. 1997, 119, 1192011925. (4) Miao, W. Chem. Rev. 2008, 108, 2506-2553. 12 ACS Paragon Plus Environment

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Figure Captions

Scheme 1. The H2S sensing mechanism of CouMC and schematic diagram of quantitative detection of H2S by measuring the max of the ECL spectra.

Figure 1. Characterization of RuSiO2@GO. (A) Representative TEM image and (B) SEM image of RuSiO2@GO. (C) The ECL spectrum of RuSiO2@GO. (D) The schematic diagram of ECL spectral setup equipped with a spectrograph CCD.

Figure 2. (A) The ECL intensity and (B) the ECL spectra from RuSiO2@GO with continuous cyclic voltammetry scans for 100 cycles.

Figure 3. (A) The overlay of the ECL spectrum of RuSiO2@GO (black solid) and the absorbance spectrum (red solid) of CouMC. (B) The overlay of the ECL spectrum of RuSiO2@GO (black solid) and the absorbance spectrum (blue solid) of CouMC in the present of H2S. (C) The ECL spectra of RuSiO2@GO film modified the carbon paper electrode in the 40 mM TPA solution upon gradual addition of CouMC (from 0 to 50 μM). (D) The simulated ECL spectra of RuSiO2@GO upon gradual addition of CouMC (from 0 to 50 μM) according to equation 1. (E) The ECL spectra obtained at the 15 ACS Paragon Plus Environment


RuSiO2@GO modified electrode in 40 mM TPA along with decreasing CouMC concentration (abbreviation: c). (F) The ECL spectra obtained at the RuSiO2@GO modified electrode in 40 mM TPA along with decreasing optical path length (abbreviation: b).

Figure 4. (A) ECL intensity responses obtained at RuSiO2@GO modified electrode in the presence of CouMC (black line) and in the presence of both CouMC and H2S (red line). (B) ECL spectra responses obtained at RuSiO2@GO modified electrode in the presence of CouMC (black line) and in the presence of both CouMC and H2S (red line). (C) The ECL intensity and the ECL spectra obtained at RuSiO2@GO modified electrodes in 20 M CouMC, 40 mM TPA and 100 mM PBS (pH = 7.4) containing 5 M and 10 M H2S, respectively. Error bars indicated s.d. (n = 5). P  0.001 (twotailed Student’s t-test). (D) The experimental ECL spectra with different ECL intensity. (E) The simulated ECL spectra with different ECL intensity. (F) The selectivity of ECL intensity and ECL spectra response towards H2S with coexisting interference agent (tetracycline, dopamine, NAD+, GSH, Cys, microcystis aeruginosa, urine and blood). Error bars indicated s.d. (n = 5). P  0.05, P  0.01, P  0.001 compared with the control group (two-tailed Student’s t-test).

Figure 5. (A) ECL intensity responses and (B) calibration curve of RuSiO2@GO film modified electrode in the 20 M CouMC and 40 mM TPA solution upon gradual addition of H2S (from 0 to 50 M). (C). ECL spectra responses and (D) calibration curve of RuSiO2@GO film modified electrode in the 20 M CouMC and 40 mM TPA solution upon gradual addition of H2S (from 0 to 50 M). Error bars indicate s.d. (n = 5).

Table 1. Standard addition recovery experiments of H2S.


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Scheme 1



Figure 1


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Figure 2



Figure 3


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RuSiO2+CouMC RuSiO2+CouMC+H2S

1000 500 0 0.6

D

0.8 1.0 Potential/V

1.2

ECL Intensity/a.u.

10000 8000 6000 4000 2000 600

650 700 Wavelength/nm

750

C

RuSiO2+CouMC RuSiO2+CouMC+H2S

1.0 0.8 0.6 0.4 0.2 0.0

600

E 8000

12000

0

1.2


750

F 400

6000

ECL Intensity/a.u.

1500

4000 2000 0

300

H2S+Tetracycline

H2S H2S+GSH

H2S+Cys

H2S+Urine

H2S+Blood

H2S+Dopamine

H2S+NAD



650 640 630



200

+

H2S+Microcystis aeruginosa

 

 

100



620 610





600


750

Figure 4


0

600 Intensity

Spectra

max/nm

B

Normalized ECL Intensity

ECL Intensity/a.u.

A 2000

ECL Intensity/a.u.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Figure 5


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Readout

Added (M)

Detected (M)

Recovery (%)

RSD (%, n = 5)

Intensity

20

9.58

47.9

3.4

𝜆𝑚𝑎𝑥

20

19.85

99.2

1.6

Table 1



for TOC only


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A Spectral Shift-Based Electrochemiluminescence Sensor for

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