Article pubs.acs.org/ac
Red−Green−Blue Electrogenerated Chemiluminescence Utilizing a Digital Camera as Detector Egan H. Doeven,† Gregory J. Barbante,† Emily Kerr,† Conor F. Hogan,*,‡ John A. Endler,§ and Paul S. Francis*,† †
Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, Victoria 3216, Australia ‡ Department of Chemistry, La Trobe Institute for Molecular Sciences, La Trobe University, Victoria 3086, Australia § Centre for Integrative Ecology, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, Victoria 3216, Australia S Supporting Information *
ABSTRACT: Exploiting the distinct excitation and emission properties of concomitant electrochemiluminophores in conjunction with the inherent color selectivity of a conventional digital camera, we create a new strategy for multiplexed electrogenerated chemiluminescence detection, suitable for the development of low-cost, portable clinical diagnostic devices. Red, green and blue emitters can be efficiently resolved over the three-dimensional space of ECL intensity versus applied potential and emission wavelength. As the relative contribution ratio of each emitter to the photographic RGB channels is constant, the RGB ECL intensity versus applied-potential curves could be effectively isolated to a single emitter at each potential.
T
electrochemiluminophores with emission maxima that span the entire visible spectrum (such as cyclometalated iridium complexes10−15) has created new opportunities for colortunable light-emitting devices16−21 and simultaneous multianalyte detection with multiple, spectrally distinct ECL species. 10,22−25 Our group recently described a novel instrumental approach whereby three-dimensional data sets comprising ECL intensity versus both emission wavelength and excitation potential can be obtained in seconds, using a combination of either cyclic voltammetry or chronoamperometry experiments with simultaneous CCD-based acquisition of the emission spectra, to provide unprecedented resolution of concomitant electrochemiluminophores.25 Although two-component mixed ECL systems have been demonstrated,10,24−26 constructing a three-color ECL system from a combination of conventional ruthenium- and/or iridium-complexes is hindered by their broad spectral distributions. The archetypal [Ru(bpy)3]2+ (orange emission) and fac-Ir(ppy)3 (green emission) complexes, for example, show considerable spectral overlap (26% of integrated peak area),10 despite the large difference in their emission maxima of 100 nm (λmax = 620 and 520 nm, respectively27). Nevertheless,
he RGB model, which describes colors in terms of three primary components, red, green, and blue, is the basis used by most modern electronic devices for the display or representation of color. The ubiquity of this technology, in particular digital cameras (such as those contained in modern mobile phones), creates an opportunity to exploit such devices for complex analytical tasks at very low cost. In this paper, we describe a new strategy for rapid multiplexed electrogenerated chemiluminescence (ECL) detection using a conventional camera as the photodetector. We demonstrate how a mixture of red, green, and blue ECL emitters within the same solution can be effectively resolved by modulating the applied potential used for excitation while simultaneously monitoring different emission wavelengths or color channels. The evolution of ECL over the past five decades from laboratory curiosity to a highly sensitive mode of detection in immunodiagnostics, life science research, and biodefense applications1−7 has been largely driven by the discovery of the light-producing reaction of tris(2,2′-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) with tri-n-propylamine (TPA) as a “coreactant” at anodic potentials in aqueous solution.8,9 In fact, [Ru(bpy)3]2+ and its close derivatives remain the only metal-complex luminophores utilized in commercial ECL systems, and therefore to perform multiple assays, the characteristic orange emission resulting from each ECL reaction must be temporally or spatially resolved using flow- or arraybased analytical formats. However, the development of © 2014 American Chemical Society
Received: December 19, 2013 Accepted: February 11, 2014 Published: February 11, 2014 2727
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
Squires (Deakin University) using a Nikon DSLR. Solutions were degassed with argon, with ECL being initiated by a series of pulses between 0 V and the specified potential. The shutter was manually activated with as close synchronization to the initiation of ECL as possible. Photographs used to investigate the limits of detection were taken using a Canon EOS 550D camera and Canon 50 mm f/1.8 lens. Identical exposure settings were used for all images (ISO 800, f/1.8). The camera shutter activation was controlled via the Autolab potentiostat configurable DIO port, using a simple transistor switch and relay to electronically control the remote shutter release (15 s exposure). ECL was initiated using a series of chronoamperometry pulses between 0 and the specified voltage. Solutions were degassed with argon for 10 min prior to analysis, but not during the data acquisition (an argon blanket was maintained for the duration). Concentrations were adjusted to achieve a balance of intensities over the emission range, as the camera has a different response curve than the corrected CCD detection. Image Analysis. Images were used directly from the camera. ImageJ (http://rsbweb.nih.gov/ij/) was used to automate the cropping and analysis of the images. Images were cropped to the smallest square area containing the emissive electrode area; with the lens, camera, and electrode used, this resulted in a 100 × 100 pixel image. The mean RGB values were measured for the circular area of the electrode using the “Measure − RGB Values” function built into ImageJ. HSV transformations of the RGB data were carried out using the following formulas:
changes in ligand structure can be exploited to control the frontier orbital energies and, therefore, the spectroscopic and electrochemical properties of the complexes.27 In this manner, we have prepared a deep red emitter ([Ru(bpy)2(dm-bpydc)]2+, where dm-bpy-dc = dimethyl 2,2′-bipyridine-4,4′dicarboxylate; λmax = 685 nm27) by introducing two electronwithdrawing methyl ester groups on one of the bpy ligands of [Ru(bpy)3]2+, which stabilized the ligand-based LUMO but left the metal-based HOMO relatively untouched, inducing a bathochromic shift in the emission while maintaining a favorable oxidation potential for ECL.27 Similarly, inclusion of fluorine groups on the phenyl rings of Ir(ppy)3 imparts a significant hypsochromic shift through stabilization of the mixed metal−ligand HOMO level, to provide a suitable blue emitter (Ir(df-ppy)3, λmax = 495 nm).24 We also prepared a complex containing a triazolylpyridinato ligand, which further blue-shifted the emission and provided a 3-fold increase in the ECL intensity (Ir(df-ppy)2(ptp), where ptp = [2-(3-phenyl-1H1,2,4-triazol-5-yl)pyridinato), λmax = 463 and 492 nm).28 Not only can these red and blue emitters be spectrally resolved (overlap in integrated peak area of only ∼5%; Figure S-1 in the Supporting Information), but here we extend this mode of detection to a third concomitant electrochemiluminophore (the spectrally overlapping green-emitter Ir(ppy)3), by exploiting the distinct potentials required for their electrochemical excitation24,25,29 and the recently discovered selective quenching of coreactant Ir(ppy)3 ECL at high overpotentials.24 Moreover, we show for the first time that the three emitters can be effectively resolved using distinct applied potentials in conjunction with the inherent color selectivity of a conventional digital camera.
■
EXPERIMENTAL SECTION Electrochemistry and ECL. Electrochemical experiments were performed with either an Autolab (Metrohm Autolab B.V., The Netherlands) PGSTAT12 (Chronoamperometry with CCD ECL detection), or PGSTAT101 (Chronoamperometry with camera detection) potentiostat. For all ECL experiments, a 3-electrode electrochemical cell was used, comprising a 3 mm glassy carbon working electrode, Ag/ AgNO3 (20 mM) (CH Instruments), nonaqueous reference electrode, and platinum wire counter electrode. Potentials are referenced to the Fc/Fc+ redox couple (measured in situ). ECL Detection. Emission spectra were captured using an Ocean Optics (Dunedin, FL, USA) QE65pro spectrometer, interfaced with our electrochemical cell using an optical fiber (1.0 m, 1.5 mm core diameter), with a collimating lens, via a custom cell holder. The electrochemical cell was located within a custom-built light-tight faraday cage for all ECL experiments. 3D-ECL emission matrix experiments were conducted under argon bubbling using the PGSTA12 potentiostat and QE65pro CCD. A series of chronoamperometry pulses for each potential point were performed, and the emissions from each group saved as a single spectrum. The integration time per spectrum was 10 s, the voltage resolution of the 3D matrix was 50 or 25 mV per spectrum (50 mV for Figure 2a, 25 mV for Figure 2b). 3D data were smoothed and graphed using SPIP analysis software. The lower coreactant ECL efficiencies of Ir(ppy)3, Ir(df-ppy)3, and Ir(df-ppy)2(ptp) were compensated for by using a larger concentration of these emitters in the mixtures used to acquire the 3D data sets. Photography. The emissions shown in Figures 3 and S-3 (Supporting Information) were photographed by Donna
■
M = max(R , G , B)
(1)
m = min(R , G , B)
(2)
C=M−m
(3)
H = 60° × H′
(4)
⎧ undefined, ⎪ ⎪ G − B mod 6, ⎪ C ⎪ H′ = ⎨ B − R + 2, ⎪ ⎪ C ⎪ R−G + 4, ⎪ ⎩ C
if C = 0 ⎫ ⎪ if M = R ⎪ ⎪ ⎪ ⎬ if M = G ⎪ ⎪ ⎪ if M = B ⎪ ⎭
(5)
RESULTS AND DISCUSSION Ideally, for the greatest selectivity, the three emitters should have different potentials for excitation. For system A ([Ru(bpy)2(dm-bpy-dc)]2+, Ir(ppy)3, and Ir(df-ppy)3), cyclic voltammetry experiments (Figure 1) showed distinct, reversible oxidation peaks emerging in the order of the green (0.33 V vs Fc0/+), blue (0.69 V) and then red (0.98 V) emitter. For system B ([Ru(bpy)2(dm-bpy-dc)]2+, Ir(ppy)3, and Ir(df-ppy)2(ptp)), the oxidative wave for the green emitter emerged first, but the oxidation peaks of the blue and red emitters were indistinguishable, and due to an as yet undefined parallel EC mechanism, the oxidative current at the potential associated with the blue emitter increased when in the presence of the green emitter. The overall mechanism leading to the emission of ECL, however, is complex and highly dependent on the nature of the coreactant;9,30 therefore, the ECL of both systems A and B were explored. 2728
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
Figure 1. Cyclic voltammogram for a mixture of (I) Ir(ppy)3, (II) Ir(df-ppy)3 and (III) [Ru(bpy)2(dm-bpy-dc)]2+ in CH3CN containing 0.1 M TBAPF6 at a scan rate of 0.1 V s−1. The concentration of each complex was 250 μM. Potentials are quoted vs Fc/Fc+. The second scan is shown. The electrochemical behavior of the individual complexes is discussed in previous publications.27,28
The ECL of [Ru(bpy)3]2+ derivatives with excess TPA as coreactant is subject to an additional parallel pathway to the excited state that involves electron transfer from the neutral TPA radical intermediate to the ruthenium complex.9 However, in the case of the two blue emitters, due to their relatively high energy LUMO levels, the TPA radical is not a sufficiently powerful reductant for an analogous light-producing pathway involving either of these complexes; (Ir(df-ppy)3: Ered = −2.51 vs Fc0/+; Ir(df-ppy)2(ptp): Ered = −2.19; compared to Ered = −1.40 for [Ru(bpy)2(dm-bpy-dc)]2+). Consequently, the red emission of light from [Ru(bpy)2(dm-bpy-dc)]2+ is observed at significantly lower potentials than its characteristic oxidation potential and the onset of ECL for both three-color systems thus occurs in the following order: green, red, and then blue, as the applied potential is increased. Three-dimensional data sets of ECL intensity versus both emission wavelength and excitation potential for these systems (Figure 2) show that the green emitter can be selectively addressed at low oxidation potentials. At moderate potentials (0.4−0.8 V vs Fc0/+), the green emitter is quenched24 and the blue emitter is not excited; therefore, only the red emitter is observed. The intensity maxima of the red and blue emitters both occur at relatively high potentials, but they remain distinguishable by their spectral distributions (Figures 2 and S-2 (Supporting Information)). From an analytical perspective, the lower ECL intensity of [Ru(bpy)2(dm-bpy-dc)]2+ at the intermediate potentials is more than compensated for by its greater ECL efficiency compared to the green and blue emitters. This instrumental approach provides an effective platform for multiplexed ECL detection and the study of electron- and energy-transfer mechanisms between luminophores and coreactants in mixed ECL systems. However, we envisage more important applications of this chemistry in ECL-based detection for paper-based microfluidic immunochemical sensors that can be operated using mobile phones (where the audio output is used to control electrochemical excitation and the in-built camera exploited as the photodetector).31,32 By harnessing the opportunities created by the rapid expansion of wireless communications networks and devices that has taken place in recent decades, mobile-phone-based sensors hold great potential to extend high-quality, low-cost clinical diagnostics,
Figure 2. 3D-ECL excitation−emission matrices for red−green−blue electrogenerated chemiluminescence (RGB-ECL) system A (3.4 μM [Ru(bpy)2(dm-bpy-dc)]2+, 165 μM Ir(ppy)3, and 85 μM Ir(df-ppy)3) and system B (1.5 μM [Ru(bpy)2(dm-bpy-dc)]2+, 150 μM Ir(ppy)3 and 20 μM Ir(df-ppy)2(ptp)). Potentials vs Fc/Fc+.
environmental monitoring and forensic testing to remote communities and the developing world.31−36 Application of multiplexed ECL detection to these devices can be achieved by a combination of selective electrochemical excitation (as described above) and photographic discrimination of emission color. Photographs of the ECL from the electrode surface at different applied potentials show a range of colors, dependent on the properties and relative concentration of electrochemiluminophores present in solution (Figures 3 and S-3 (Supporting Information)). In agreement with the spectroscopic data (Figures 2 and S-2 (Supporting Information)), at
Figure 3. Photographs of the ECL at the 3 mm diameter working electrode surface at different applied potentials. Conditions: 10 mM TPA coreactant and 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) electrolyte, dissolved in acetonitrile. An identical exposure time was used for each image. Potentials vs Fc/Fc+. 2729
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
low potentials, only the green emission from Ir(ppy)3 was observed. At intermediate potentials (∼0.7 V), the red emitter was detected, albeit not at its maximum intensity. At relatively high potentials, both the red and blue emitters were activated, which was seen as a magenta emission from the mixed electrochemiluminophore solution. From a color-tuning standpoint, a wide range of other colors can be generated by manipulating the spectral distribution or the concentrations of the emitters. With respect to multiplexed ECL, the selective excitation and detection of electrochemiluminophores (Figures 2 and S-2 (Supporting Information)) enables discrimination of the three components by a combination of wavelength and potential resolved measurements, after calibration for electrochemical interactions between the concomitant ECL systems. Although this is certainly attractive for multiplexed analysis, the capacity to distinguish between different colored emitters (at each applied potential) using simple camera-based detection vastly expands the potential applicability of this approach for applications such as low-cost point of care diagnostics, by allowing this extra dimension of selectivity without recourse to ancillary optics or expensive detectors. As a demonstration of the use of digital cameras to discriminate between electrochemiluminophores, we analyzed the photographs of ECL (Figures 3 and S-3 (Supporting Information)) using ImageJ software to obtain mean RGB values for the emission at each applied potential. Due to their broad spectral distribution, the “blue” emission from either Ir(df-ppy)2(ptp) or Ir(df-ppy)3 was found to contribute to both the B and G channels and the “green” emission from Ir(ppy)3 contributed to all three channels (Figure 4). The “red” emission from [Ru(bpy)2(dm-bpy-dc)]2+, however, was confined to the R channel. Thus, using a combination of the inherent selectivity associated with the electrochemical excitation and the photonic emission, each emitter could be measured at its maximum intensity. Therefore, as shown in Figure 4, at applied potentials of 0.25 and 0.8 V, respectively, where the green and red emitters are selectively excited (Table 1), the sum of the intensities of the three channels may be used for quantitation. At high potentials, on the other hand, the simultaneously emitting blue and red emitters can be selectively measured via the B and R channels, respectively. As the contribution ratio of each emitter to the RGB channels was constant, the resulting RGB ECL intensity versus applied-potential curves (obtained using the camera as ECL photodetector) at all potentials could be isolated to a single emitter (Figure 4b), suggesting that there is great opportunity to expand this approach to higher numbers of concomitant emitters by deconvolution of their respective contributions to the three basic color channels at multiple excitation potentials. Using this approach, the analytical working range can be conveniently adjusted by setting the exposure time of the camera. In this demonstration, a 15 s exposure time was employed, and the limits of detection for the red, blue and green emitters were 0.07, 0.2, and 0.4 μM. Under these chemical and instrumental conditions, the analytical working range extended up to 15, 30, and 100 μM (Figure S-4, Supporting Information) and the calibration equations were the following: y = (−1.9 × 105)x2 + (9.1 × 103)x, y = (−4.7 × 104) x2 + (5.6 × 103)x, and y = (−4.2 × 103)x2 + (8.7 × 102)x, with R2 values of 0.9958, 0.9944, and 0.9997, respectively. Considerable scope exists for achieving lower detection limits
Figure 4. (a) ECL intensity in the R, G, and B channels vs applied potential for a mixture of Ir(ppy)3 (green emitter, 25 μM), [Ru(bpy)2(dm-bpy-dc)]2+ (red emitter, 3.75 μM), and Ir(dfppy)2(ptp) (blue emitter, 7.5 μM). (b) After correction for the contribution of the green emitter to the R and B channels and the contribution of the blue emitter to the G channel. Potential vs Fc/Fc+.
Table 1. Contribution of Each Emitter to the R, G, and B Channels for System B RGB channel potential/V
red
0.3 0.7 1.0
Ir(ppy)3 [Ru(bpy)2(L)]2+ [Ru(bpy)2(L)]2+
green
blue
Ir(ppy)3
Ir(ppy)3
Ir(df-ppy)2(ptp)
Ir(df-ppy)2(ptp)
by further optimizing the detection parameters and the chemistry of the ECL systems. The RGB approach for ECL measurement also enables other well-established treatments of color data to be exploited. Figure 5 shows a HSV (Hue, Saturation, Value) representation of the color changes observed during an ECL experiment with the three luminophores. Such a transformation of the RGB data is advantageous because it allows the color information to be represented by a single parameter: the coordinate H (hue).37−39 This figure illustrates how both qualitative and quantitative information may be obtained by monitoring changes in emission color (H coordinate) and emission intensity (V coordinate). On the basis of these proof-of-concept findings, we are currently developing new metal complexes to further improve the sensitivity and selectivity of this ECL approach, and exploring the application of this technology to multiplexed immunochemical assays within portable, paper-based microfluidic devices. 2730
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
*Paul S. Francis: E-mail: [email protected]. Phone: +61 3 5227 1294. Fax: +61 3 5227 2356. Author Contributions
The paper was written through contributions of all authors. All authors have given approval to the final version of the paper. Funding
This work was funded by the Australian Research Council (FT100100646, LE120100213, DP1094179). Notes
The authors declare no competing financial interest.
■
(1) Bard, A. J. Electrogenerated Chemiluminescence; Marcel Dekker: New York, 2004. (2) Richter, M. M. Chem. Rev. 2004, 104, 3003−3036. (3) Gorman, B. A.; Francis, P. S.; Barnett, N. W. Analyst 2006, 131, 616−639. (4) Miao, W. Chem. Rev. 2008, 108, 2506−2553. (5) Forster, R. J.; Bertoncello, P.; Keyes, T. E. Annu. Rev. Anal. Chem. 2009, 2, 359−385. (6) Hu, L.; Xu, G. Chem. Soc. Rev. 2010, 39, 3275−3304. (7) Forster, R. J.; Keyes, T. E. Neuromethods 2013, 80, 347−367. (8) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 3127− 3131. (9) Miao, W.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478−14485. (10) Bruce, D.; Richter, M. M. Anal. Chem. 2002, 74, 1340−1342. (11) Kim, J. I.; Shin, I.-S.; Kim, H.; Lee, J.-K. J. Am. Chem. Soc. 2005, 127, 1614−1615. (12) Zanarini, S.; Rampazzo, E.; Bonacchi, S.; Juris, R.; Marcaccio, M.; Montalti, M.; Paolucci, F.; Prodi, L. J. Am. Chem. Soc. 2009, 131, 14208−14209. (13) Zanarini, S.; Felici, M.; Valenti, G.; Marcaccio, M.; Prodi, L.; Bonacchi, S.; Contreras-Carballada, P.; Williams, R. M.; Feiters, M. C.; Nolte, R. J. M.; De Cola, L.; Paolucci, F. Chem.−Eur. J. 2011, 17, 4640−4647. (14) Swanick, K. N.; Ladouceur, S.; Zysman-Colman, E.; Ding, Z. Angew. Chem., Int. Ed. 2012, 51, 11079−11082. (15) Kiran, R. V.; Hogan, C. F.; James, B. D.; Wilson, D. J. D. Eur. J. Inorg. Chem. 2011, 4816−4825. (16) Gao, F. G.; Bard, A. J. J. Am. Chem. Soc. 2000, 122, 7426−7427. (17) Welter, S.; Brunner, K.; Hofstraat, J. W.; De Cola, L. Nature 2003, 421, 54−57. (18) Slinker Jason, D.; Gorodetsky Alon, A.; Lowry Michael, S.; Wang, J.; Parker, S.; Rohl, R.; Bernhard, S.; Malliaras George, G. J. Am. Chem. Soc. 2004, 126, 2763−2767. (19) Wang, F.; Wang, P.; Fan, X.; Dang, X.; Zhen, C.; Zou, D.; Kim, E. H.; Lee, D. N.; Kim, B. H. Appl. Phys. Lett. 2006, 89, 183519/ 183511−183519/183513. (20) Su, H.-C.; Chen, H.-F.; Fang, F.-C.; Liu, C.-C.; Wu, C.-C.; Wong, K.-T.; Liu, Y.-H.; Peng, S.-M. J. Am. Chem. Soc. 2008, 130, 3413−3419. (21) He, L.; Duan, L.; Qiao, J.; Wang, R.; Wei, P.; Wang, L.; Qiu, Y. Adv. Funct. Mater. 2008, 18, 2123−2131. (22) Muegge, B. D.; Richter, M. M. Luminescence 2005, 20, 76−80. (23) Guo, Z.; Hao, T.; Du, S.; Chen, B.; Wang, Z.; Li, X.; Wang, S. Biosens. Bioelectron. 2013, 44, 101−107. (24) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Francis, P. S.; Barnett, N. W.; Hogan, C. F. Chem. Sci. 2013, 4, 977−982. (25) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Hogan, C. F.; Barnett, N. W.; Francis, P. S. Angew. Chem., Int. Ed. 2012, 51, 4354− 4357. (26) Muegge, B. D.; Richter, M. M. Anal. Chem. 2004, 76, 73−77. (27) Barbante, G. J.; Hogan, C. F.; Wilson, D. J. D.; Lewcenko, N. A.; Pfeffer, F. M.; Barnett, N. W.; Francis, P. S. Analyst 2011, 136, 1329− 1338.
Figure 5. HSV representation of spectral changes observed during ECL experiment with three luminophores, characterizing the change in observed color with potential (as in Figure S-3 (Supporting Information), bottom row). Values of H (hue) and V (value) were derived from photographs of the working electrode surface obtained at different applied potentials (every 50 mV, from 0.17 to 1.22 V vs Fc/ Fc+) for a mixture of Ir(ppy)3 (green emitter), [Ru(bpy)2(dm-bpydc)]2+ (red emitter), and Ir(df-ppy)2(ptp) (blue emitter). Value (V) (in the top graph) is indicative of the overall intensity of the emission, whereas the hue (H) is a numerical representation of the color expressed as an angular coordinate. The positions of green, blue and red emitter standards on this scale, when measured separately, were 87°, 180°, and 360°, respectively.
■
ASSOCIATED CONTENT
S Supporting Information *
Chemical structures and individual ECL spectra for the complexes used in Systems A and B, ECL spectra extracted from Figure 2B at different applied potentials, and additional photographs of the ECL at the working electrode surface at different applied potentials. This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Conor F. Hogan: E-mail: [email protected]. Phone: +61 3 9479 3747. Fax: +61 3 9479 1399. 2731
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
(28) Barbante, G. J.; Doeven, E. H.; Kerr, E.; Connell, T. U.; Donnelly, P. S.; White, J. M.; Lópes, T.; Laird, S.; Hogan, C. F.; Wilson, D. J. D.; Barnard, P. J.; Francis, P. S. Chem.−Eur. J. 2014, DOI: 10.1002/chem.201304500. (29) Wang, S.; Ge, L.; Zhang, Y.; Song, X.; Li, N.; Ge, S.; Yu, J. Lab Chip 2012, 12, 4489−4498. (30) Liu, X.; Shi, L.; Niu, W.; Li, H.; Xu, G. Angew. Chem., Int. Ed. 2007, 46, 421−424. (31) Delaney, J. L.; Hogan, C. F.; Tian, J.; Shen, W. Anal. Chem. 2011, 83, 1300−1306. (32) Delaney, J. L.; Doeven, E. H.; Harsant, A. J.; Hogan, C. F. Anal. Chim. Acta 2013, 790, 56−60. (33) Lillehoj, P. B.; Huang, M.-C.; Truong, N.; Ho, C.-M. Lab Chip 2013, 13, 2950−2955. (34) Zhu, H.-Y.; Mavandadi, S.; Coskun, A. F.; Yaglidere, O.; Ozcan, A. Anal. Chem. 2011, 83, 6641−6647. (35) Martinez, A. W.; Phillips, S. T.; Carrilho, E.; Thomas, S. W.; Sindi, H.; Whitesides, G. M. Anal. Chem. 2008, 80, 3699−3707. (36) Mudanyali, O.; Dimitrov, S.; Sikora, U.; Padmanabhan, S.; Navruz, I.; Ozcan, A. Lab Chip 2012, 12, 2678−2686. (37) Chang, B.-Y. Bull. Korean Chem. Soc. 2012, 33, 549−552. (38) Oncescu, V.; O’Dell, D.; Erickson, D. Lab Chip 2013, 13, 3232− 3238. (39) Oncescu, V.; Mancuso, M.; Erickson, D. Lab Chip, DOI: 10.1039/c1033lc51194d.
2732
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Red−Green−Blue Electrogenerated Chemiluminescence Utilizing a Digital Camera as Detector Egan H. Doeven,† Gregory J. Barbante,† Emily Kerr,† Conor F. Hogan,*,‡ John A. Endler,§ and Paul S. Francis*,† †
Centre for Chemistry and Biotechnology, School of Life and Environmental Sciences, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, Victoria 3216, Australia ‡ Department of Chemistry, La Trobe Institute for Molecular Sciences, La Trobe University, Victoria 3086, Australia § Centre for Integrative Ecology, Faculty of Science, Engineering and Built Environment, Deakin University, Waurn Ponds, Victoria 3216, Australia S Supporting Information *
ABSTRACT: Exploiting the distinct excitation and emission properties of concomitant electrochemiluminophores in conjunction with the inherent color selectivity of a conventional digital camera, we create a new strategy for multiplexed electrogenerated chemiluminescence detection, suitable for the development of low-cost, portable clinical diagnostic devices. Red, green and blue emitters can be efficiently resolved over the three-dimensional space of ECL intensity versus applied potential and emission wavelength. As the relative contribution ratio of each emitter to the photographic RGB channels is constant, the RGB ECL intensity versus applied-potential curves could be effectively isolated to a single emitter at each potential.
T
electrochemiluminophores with emission maxima that span the entire visible spectrum (such as cyclometalated iridium complexes10−15) has created new opportunities for colortunable light-emitting devices16−21 and simultaneous multianalyte detection with multiple, spectrally distinct ECL species. 10,22−25 Our group recently described a novel instrumental approach whereby three-dimensional data sets comprising ECL intensity versus both emission wavelength and excitation potential can be obtained in seconds, using a combination of either cyclic voltammetry or chronoamperometry experiments with simultaneous CCD-based acquisition of the emission spectra, to provide unprecedented resolution of concomitant electrochemiluminophores.25 Although two-component mixed ECL systems have been demonstrated,10,24−26 constructing a three-color ECL system from a combination of conventional ruthenium- and/or iridium-complexes is hindered by their broad spectral distributions. The archetypal [Ru(bpy)3]2+ (orange emission) and fac-Ir(ppy)3 (green emission) complexes, for example, show considerable spectral overlap (26% of integrated peak area),10 despite the large difference in their emission maxima of 100 nm (λmax = 620 and 520 nm, respectively27). Nevertheless,
he RGB model, which describes colors in terms of three primary components, red, green, and blue, is the basis used by most modern electronic devices for the display or representation of color. The ubiquity of this technology, in particular digital cameras (such as those contained in modern mobile phones), creates an opportunity to exploit such devices for complex analytical tasks at very low cost. In this paper, we describe a new strategy for rapid multiplexed electrogenerated chemiluminescence (ECL) detection using a conventional camera as the photodetector. We demonstrate how a mixture of red, green, and blue ECL emitters within the same solution can be effectively resolved by modulating the applied potential used for excitation while simultaneously monitoring different emission wavelengths or color channels. The evolution of ECL over the past five decades from laboratory curiosity to a highly sensitive mode of detection in immunodiagnostics, life science research, and biodefense applications1−7 has been largely driven by the discovery of the light-producing reaction of tris(2,2′-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) with tri-n-propylamine (TPA) as a “coreactant” at anodic potentials in aqueous solution.8,9 In fact, [Ru(bpy)3]2+ and its close derivatives remain the only metal-complex luminophores utilized in commercial ECL systems, and therefore to perform multiple assays, the characteristic orange emission resulting from each ECL reaction must be temporally or spatially resolved using flow- or arraybased analytical formats. However, the development of © 2014 American Chemical Society
Received: December 19, 2013 Accepted: February 11, 2014 Published: February 11, 2014 2727
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
Squires (Deakin University) using a Nikon DSLR. Solutions were degassed with argon, with ECL being initiated by a series of pulses between 0 V and the specified potential. The shutter was manually activated with as close synchronization to the initiation of ECL as possible. Photographs used to investigate the limits of detection were taken using a Canon EOS 550D camera and Canon 50 mm f/1.8 lens. Identical exposure settings were used for all images (ISO 800, f/1.8). The camera shutter activation was controlled via the Autolab potentiostat configurable DIO port, using a simple transistor switch and relay to electronically control the remote shutter release (15 s exposure). ECL was initiated using a series of chronoamperometry pulses between 0 and the specified voltage. Solutions were degassed with argon for 10 min prior to analysis, but not during the data acquisition (an argon blanket was maintained for the duration). Concentrations were adjusted to achieve a balance of intensities over the emission range, as the camera has a different response curve than the corrected CCD detection. Image Analysis. Images were used directly from the camera. ImageJ (http://rsbweb.nih.gov/ij/) was used to automate the cropping and analysis of the images. Images were cropped to the smallest square area containing the emissive electrode area; with the lens, camera, and electrode used, this resulted in a 100 × 100 pixel image. The mean RGB values were measured for the circular area of the electrode using the “Measure − RGB Values” function built into ImageJ. HSV transformations of the RGB data were carried out using the following formulas:
changes in ligand structure can be exploited to control the frontier orbital energies and, therefore, the spectroscopic and electrochemical properties of the complexes.27 In this manner, we have prepared a deep red emitter ([Ru(bpy)2(dm-bpydc)]2+, where dm-bpy-dc = dimethyl 2,2′-bipyridine-4,4′dicarboxylate; λmax = 685 nm27) by introducing two electronwithdrawing methyl ester groups on one of the bpy ligands of [Ru(bpy)3]2+, which stabilized the ligand-based LUMO but left the metal-based HOMO relatively untouched, inducing a bathochromic shift in the emission while maintaining a favorable oxidation potential for ECL.27 Similarly, inclusion of fluorine groups on the phenyl rings of Ir(ppy)3 imparts a significant hypsochromic shift through stabilization of the mixed metal−ligand HOMO level, to provide a suitable blue emitter (Ir(df-ppy)3, λmax = 495 nm).24 We also prepared a complex containing a triazolylpyridinato ligand, which further blue-shifted the emission and provided a 3-fold increase in the ECL intensity (Ir(df-ppy)2(ptp), where ptp = [2-(3-phenyl-1H1,2,4-triazol-5-yl)pyridinato), λmax = 463 and 492 nm).28 Not only can these red and blue emitters be spectrally resolved (overlap in integrated peak area of only ∼5%; Figure S-1 in the Supporting Information), but here we extend this mode of detection to a third concomitant electrochemiluminophore (the spectrally overlapping green-emitter Ir(ppy)3), by exploiting the distinct potentials required for their electrochemical excitation24,25,29 and the recently discovered selective quenching of coreactant Ir(ppy)3 ECL at high overpotentials.24 Moreover, we show for the first time that the three emitters can be effectively resolved using distinct applied potentials in conjunction with the inherent color selectivity of a conventional digital camera.
■
EXPERIMENTAL SECTION Electrochemistry and ECL. Electrochemical experiments were performed with either an Autolab (Metrohm Autolab B.V., The Netherlands) PGSTAT12 (Chronoamperometry with CCD ECL detection), or PGSTAT101 (Chronoamperometry with camera detection) potentiostat. For all ECL experiments, a 3-electrode electrochemical cell was used, comprising a 3 mm glassy carbon working electrode, Ag/ AgNO3 (20 mM) (CH Instruments), nonaqueous reference electrode, and platinum wire counter electrode. Potentials are referenced to the Fc/Fc+ redox couple (measured in situ). ECL Detection. Emission spectra were captured using an Ocean Optics (Dunedin, FL, USA) QE65pro spectrometer, interfaced with our electrochemical cell using an optical fiber (1.0 m, 1.5 mm core diameter), with a collimating lens, via a custom cell holder. The electrochemical cell was located within a custom-built light-tight faraday cage for all ECL experiments. 3D-ECL emission matrix experiments were conducted under argon bubbling using the PGSTA12 potentiostat and QE65pro CCD. A series of chronoamperometry pulses for each potential point were performed, and the emissions from each group saved as a single spectrum. The integration time per spectrum was 10 s, the voltage resolution of the 3D matrix was 50 or 25 mV per spectrum (50 mV for Figure 2a, 25 mV for Figure 2b). 3D data were smoothed and graphed using SPIP analysis software. The lower coreactant ECL efficiencies of Ir(ppy)3, Ir(df-ppy)3, and Ir(df-ppy)2(ptp) were compensated for by using a larger concentration of these emitters in the mixtures used to acquire the 3D data sets. Photography. The emissions shown in Figures 3 and S-3 (Supporting Information) were photographed by Donna
■
M = max(R , G , B)
(1)
m = min(R , G , B)
(2)
C=M−m
(3)
H = 60° × H′
(4)
⎧ undefined, ⎪ ⎪ G − B mod 6, ⎪ C ⎪ H′ = ⎨ B − R + 2, ⎪ ⎪ C ⎪ R−G + 4, ⎪ ⎩ C
if C = 0 ⎫ ⎪ if M = R ⎪ ⎪ ⎪ ⎬ if M = G ⎪ ⎪ ⎪ if M = B ⎪ ⎭
(5)
RESULTS AND DISCUSSION Ideally, for the greatest selectivity, the three emitters should have different potentials for excitation. For system A ([Ru(bpy)2(dm-bpy-dc)]2+, Ir(ppy)3, and Ir(df-ppy)3), cyclic voltammetry experiments (Figure 1) showed distinct, reversible oxidation peaks emerging in the order of the green (0.33 V vs Fc0/+), blue (0.69 V) and then red (0.98 V) emitter. For system B ([Ru(bpy)2(dm-bpy-dc)]2+, Ir(ppy)3, and Ir(df-ppy)2(ptp)), the oxidative wave for the green emitter emerged first, but the oxidation peaks of the blue and red emitters were indistinguishable, and due to an as yet undefined parallel EC mechanism, the oxidative current at the potential associated with the blue emitter increased when in the presence of the green emitter. The overall mechanism leading to the emission of ECL, however, is complex and highly dependent on the nature of the coreactant;9,30 therefore, the ECL of both systems A and B were explored. 2728
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
Figure 1. Cyclic voltammogram for a mixture of (I) Ir(ppy)3, (II) Ir(df-ppy)3 and (III) [Ru(bpy)2(dm-bpy-dc)]2+ in CH3CN containing 0.1 M TBAPF6 at a scan rate of 0.1 V s−1. The concentration of each complex was 250 μM. Potentials are quoted vs Fc/Fc+. The second scan is shown. The electrochemical behavior of the individual complexes is discussed in previous publications.27,28
The ECL of [Ru(bpy)3]2+ derivatives with excess TPA as coreactant is subject to an additional parallel pathway to the excited state that involves electron transfer from the neutral TPA radical intermediate to the ruthenium complex.9 However, in the case of the two blue emitters, due to their relatively high energy LUMO levels, the TPA radical is not a sufficiently powerful reductant for an analogous light-producing pathway involving either of these complexes; (Ir(df-ppy)3: Ered = −2.51 vs Fc0/+; Ir(df-ppy)2(ptp): Ered = −2.19; compared to Ered = −1.40 for [Ru(bpy)2(dm-bpy-dc)]2+). Consequently, the red emission of light from [Ru(bpy)2(dm-bpy-dc)]2+ is observed at significantly lower potentials than its characteristic oxidation potential and the onset of ECL for both three-color systems thus occurs in the following order: green, red, and then blue, as the applied potential is increased. Three-dimensional data sets of ECL intensity versus both emission wavelength and excitation potential for these systems (Figure 2) show that the green emitter can be selectively addressed at low oxidation potentials. At moderate potentials (0.4−0.8 V vs Fc0/+), the green emitter is quenched24 and the blue emitter is not excited; therefore, only the red emitter is observed. The intensity maxima of the red and blue emitters both occur at relatively high potentials, but they remain distinguishable by their spectral distributions (Figures 2 and S-2 (Supporting Information)). From an analytical perspective, the lower ECL intensity of [Ru(bpy)2(dm-bpy-dc)]2+ at the intermediate potentials is more than compensated for by its greater ECL efficiency compared to the green and blue emitters. This instrumental approach provides an effective platform for multiplexed ECL detection and the study of electron- and energy-transfer mechanisms between luminophores and coreactants in mixed ECL systems. However, we envisage more important applications of this chemistry in ECL-based detection for paper-based microfluidic immunochemical sensors that can be operated using mobile phones (where the audio output is used to control electrochemical excitation and the in-built camera exploited as the photodetector).31,32 By harnessing the opportunities created by the rapid expansion of wireless communications networks and devices that has taken place in recent decades, mobile-phone-based sensors hold great potential to extend high-quality, low-cost clinical diagnostics,
Figure 2. 3D-ECL excitation−emission matrices for red−green−blue electrogenerated chemiluminescence (RGB-ECL) system A (3.4 μM [Ru(bpy)2(dm-bpy-dc)]2+, 165 μM Ir(ppy)3, and 85 μM Ir(df-ppy)3) and system B (1.5 μM [Ru(bpy)2(dm-bpy-dc)]2+, 150 μM Ir(ppy)3 and 20 μM Ir(df-ppy)2(ptp)). Potentials vs Fc/Fc+.
environmental monitoring and forensic testing to remote communities and the developing world.31−36 Application of multiplexed ECL detection to these devices can be achieved by a combination of selective electrochemical excitation (as described above) and photographic discrimination of emission color. Photographs of the ECL from the electrode surface at different applied potentials show a range of colors, dependent on the properties and relative concentration of electrochemiluminophores present in solution (Figures 3 and S-3 (Supporting Information)). In agreement with the spectroscopic data (Figures 2 and S-2 (Supporting Information)), at
Figure 3. Photographs of the ECL at the 3 mm diameter working electrode surface at different applied potentials. Conditions: 10 mM TPA coreactant and 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6) electrolyte, dissolved in acetonitrile. An identical exposure time was used for each image. Potentials vs Fc/Fc+. 2729
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
low potentials, only the green emission from Ir(ppy)3 was observed. At intermediate potentials (∼0.7 V), the red emitter was detected, albeit not at its maximum intensity. At relatively high potentials, both the red and blue emitters were activated, which was seen as a magenta emission from the mixed electrochemiluminophore solution. From a color-tuning standpoint, a wide range of other colors can be generated by manipulating the spectral distribution or the concentrations of the emitters. With respect to multiplexed ECL, the selective excitation and detection of electrochemiluminophores (Figures 2 and S-2 (Supporting Information)) enables discrimination of the three components by a combination of wavelength and potential resolved measurements, after calibration for electrochemical interactions between the concomitant ECL systems. Although this is certainly attractive for multiplexed analysis, the capacity to distinguish between different colored emitters (at each applied potential) using simple camera-based detection vastly expands the potential applicability of this approach for applications such as low-cost point of care diagnostics, by allowing this extra dimension of selectivity without recourse to ancillary optics or expensive detectors. As a demonstration of the use of digital cameras to discriminate between electrochemiluminophores, we analyzed the photographs of ECL (Figures 3 and S-3 (Supporting Information)) using ImageJ software to obtain mean RGB values for the emission at each applied potential. Due to their broad spectral distribution, the “blue” emission from either Ir(df-ppy)2(ptp) or Ir(df-ppy)3 was found to contribute to both the B and G channels and the “green” emission from Ir(ppy)3 contributed to all three channels (Figure 4). The “red” emission from [Ru(bpy)2(dm-bpy-dc)]2+, however, was confined to the R channel. Thus, using a combination of the inherent selectivity associated with the electrochemical excitation and the photonic emission, each emitter could be measured at its maximum intensity. Therefore, as shown in Figure 4, at applied potentials of 0.25 and 0.8 V, respectively, where the green and red emitters are selectively excited (Table 1), the sum of the intensities of the three channels may be used for quantitation. At high potentials, on the other hand, the simultaneously emitting blue and red emitters can be selectively measured via the B and R channels, respectively. As the contribution ratio of each emitter to the RGB channels was constant, the resulting RGB ECL intensity versus applied-potential curves (obtained using the camera as ECL photodetector) at all potentials could be isolated to a single emitter (Figure 4b), suggesting that there is great opportunity to expand this approach to higher numbers of concomitant emitters by deconvolution of their respective contributions to the three basic color channels at multiple excitation potentials. Using this approach, the analytical working range can be conveniently adjusted by setting the exposure time of the camera. In this demonstration, a 15 s exposure time was employed, and the limits of detection for the red, blue and green emitters were 0.07, 0.2, and 0.4 μM. Under these chemical and instrumental conditions, the analytical working range extended up to 15, 30, and 100 μM (Figure S-4, Supporting Information) and the calibration equations were the following: y = (−1.9 × 105)x2 + (9.1 × 103)x, y = (−4.7 × 104) x2 + (5.6 × 103)x, and y = (−4.2 × 103)x2 + (8.7 × 102)x, with R2 values of 0.9958, 0.9944, and 0.9997, respectively. Considerable scope exists for achieving lower detection limits
Figure 4. (a) ECL intensity in the R, G, and B channels vs applied potential for a mixture of Ir(ppy)3 (green emitter, 25 μM), [Ru(bpy)2(dm-bpy-dc)]2+ (red emitter, 3.75 μM), and Ir(dfppy)2(ptp) (blue emitter, 7.5 μM). (b) After correction for the contribution of the green emitter to the R and B channels and the contribution of the blue emitter to the G channel. Potential vs Fc/Fc+.
Table 1. Contribution of Each Emitter to the R, G, and B Channels for System B RGB channel potential/V
red
0.3 0.7 1.0
Ir(ppy)3 [Ru(bpy)2(L)]2+ [Ru(bpy)2(L)]2+
green
blue
Ir(ppy)3
Ir(ppy)3
Ir(df-ppy)2(ptp)
Ir(df-ppy)2(ptp)
by further optimizing the detection parameters and the chemistry of the ECL systems. The RGB approach for ECL measurement also enables other well-established treatments of color data to be exploited. Figure 5 shows a HSV (Hue, Saturation, Value) representation of the color changes observed during an ECL experiment with the three luminophores. Such a transformation of the RGB data is advantageous because it allows the color information to be represented by a single parameter: the coordinate H (hue).37−39 This figure illustrates how both qualitative and quantitative information may be obtained by monitoring changes in emission color (H coordinate) and emission intensity (V coordinate). On the basis of these proof-of-concept findings, we are currently developing new metal complexes to further improve the sensitivity and selectivity of this ECL approach, and exploring the application of this technology to multiplexed immunochemical assays within portable, paper-based microfluidic devices. 2730
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
*Paul S. Francis: E-mail: [email protected]. Phone: +61 3 5227 1294. Fax: +61 3 5227 2356. Author Contributions
The paper was written through contributions of all authors. All authors have given approval to the final version of the paper. Funding
This work was funded by the Australian Research Council (FT100100646, LE120100213, DP1094179). Notes
The authors declare no competing financial interest.
■
(1) Bard, A. J. Electrogenerated Chemiluminescence; Marcel Dekker: New York, 2004. (2) Richter, M. M. Chem. Rev. 2004, 104, 3003−3036. (3) Gorman, B. A.; Francis, P. S.; Barnett, N. W. Analyst 2006, 131, 616−639. (4) Miao, W. Chem. Rev. 2008, 108, 2506−2553. (5) Forster, R. J.; Bertoncello, P.; Keyes, T. E. Annu. Rev. Anal. Chem. 2009, 2, 359−385. (6) Hu, L.; Xu, G. Chem. Soc. Rev. 2010, 39, 3275−3304. (7) Forster, R. J.; Keyes, T. E. Neuromethods 2013, 80, 347−367. (8) Leland, J. K.; Powell, M. J. J. Electrochem. Soc. 1990, 137, 3127− 3131. (9) Miao, W.; Choi, J.-P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124, 14478−14485. (10) Bruce, D.; Richter, M. M. Anal. Chem. 2002, 74, 1340−1342. (11) Kim, J. I.; Shin, I.-S.; Kim, H.; Lee, J.-K. J. Am. Chem. Soc. 2005, 127, 1614−1615. (12) Zanarini, S.; Rampazzo, E.; Bonacchi, S.; Juris, R.; Marcaccio, M.; Montalti, M.; Paolucci, F.; Prodi, L. J. Am. Chem. Soc. 2009, 131, 14208−14209. (13) Zanarini, S.; Felici, M.; Valenti, G.; Marcaccio, M.; Prodi, L.; Bonacchi, S.; Contreras-Carballada, P.; Williams, R. M.; Feiters, M. C.; Nolte, R. J. M.; De Cola, L.; Paolucci, F. Chem.−Eur. J. 2011, 17, 4640−4647. (14) Swanick, K. N.; Ladouceur, S.; Zysman-Colman, E.; Ding, Z. Angew. Chem., Int. Ed. 2012, 51, 11079−11082. (15) Kiran, R. V.; Hogan, C. F.; James, B. D.; Wilson, D. J. D. Eur. J. Inorg. Chem. 2011, 4816−4825. (16) Gao, F. G.; Bard, A. J. J. Am. Chem. Soc. 2000, 122, 7426−7427. (17) Welter, S.; Brunner, K.; Hofstraat, J. W.; De Cola, L. Nature 2003, 421, 54−57. (18) Slinker Jason, D.; Gorodetsky Alon, A.; Lowry Michael, S.; Wang, J.; Parker, S.; Rohl, R.; Bernhard, S.; Malliaras George, G. J. Am. Chem. Soc. 2004, 126, 2763−2767. (19) Wang, F.; Wang, P.; Fan, X.; Dang, X.; Zhen, C.; Zou, D.; Kim, E. H.; Lee, D. N.; Kim, B. H. Appl. Phys. Lett. 2006, 89, 183519/ 183511−183519/183513. (20) Su, H.-C.; Chen, H.-F.; Fang, F.-C.; Liu, C.-C.; Wu, C.-C.; Wong, K.-T.; Liu, Y.-H.; Peng, S.-M. J. Am. Chem. Soc. 2008, 130, 3413−3419. (21) He, L.; Duan, L.; Qiao, J.; Wang, R.; Wei, P.; Wang, L.; Qiu, Y. Adv. Funct. Mater. 2008, 18, 2123−2131. (22) Muegge, B. D.; Richter, M. M. Luminescence 2005, 20, 76−80. (23) Guo, Z.; Hao, T.; Du, S.; Chen, B.; Wang, Z.; Li, X.; Wang, S. Biosens. Bioelectron. 2013, 44, 101−107. (24) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Francis, P. S.; Barnett, N. W.; Hogan, C. F. Chem. Sci. 2013, 4, 977−982. (25) Doeven, E. H.; Zammit, E. M.; Barbante, G. J.; Hogan, C. F.; Barnett, N. W.; Francis, P. S. Angew. Chem., Int. Ed. 2012, 51, 4354− 4357. (26) Muegge, B. D.; Richter, M. M. Anal. Chem. 2004, 76, 73−77. (27) Barbante, G. J.; Hogan, C. F.; Wilson, D. J. D.; Lewcenko, N. A.; Pfeffer, F. M.; Barnett, N. W.; Francis, P. S. Analyst 2011, 136, 1329− 1338.
Figure 5. HSV representation of spectral changes observed during ECL experiment with three luminophores, characterizing the change in observed color with potential (as in Figure S-3 (Supporting Information), bottom row). Values of H (hue) and V (value) were derived from photographs of the working electrode surface obtained at different applied potentials (every 50 mV, from 0.17 to 1.22 V vs Fc/ Fc+) for a mixture of Ir(ppy)3 (green emitter), [Ru(bpy)2(dm-bpydc)]2+ (red emitter), and Ir(df-ppy)2(ptp) (blue emitter). Value (V) (in the top graph) is indicative of the overall intensity of the emission, whereas the hue (H) is a numerical representation of the color expressed as an angular coordinate. The positions of green, blue and red emitter standards on this scale, when measured separately, were 87°, 180°, and 360°, respectively.
■
ASSOCIATED CONTENT
S Supporting Information *
Chemical structures and individual ECL spectra for the complexes used in Systems A and B, ECL spectra extracted from Figure 2B at different applied potentials, and additional photographs of the ECL at the working electrode surface at different applied potentials. This material is available free of charge via the Internet at http://pubs.acs.org.
■
REFERENCES
AUTHOR INFORMATION
Corresponding Authors
*Conor F. Hogan: E-mail: [email protected]. Phone: +61 3 9479 3747. Fax: +61 3 9479 1399. 2731
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
Analytical Chemistry
Article
(28) Barbante, G. J.; Doeven, E. H.; Kerr, E.; Connell, T. U.; Donnelly, P. S.; White, J. M.; Lópes, T.; Laird, S.; Hogan, C. F.; Wilson, D. J. D.; Barnard, P. J.; Francis, P. S. Chem.−Eur. J. 2014, DOI: 10.1002/chem.201304500. (29) Wang, S.; Ge, L.; Zhang, Y.; Song, X.; Li, N.; Ge, S.; Yu, J. Lab Chip 2012, 12, 4489−4498. (30) Liu, X.; Shi, L.; Niu, W.; Li, H.; Xu, G. Angew. Chem., Int. Ed. 2007, 46, 421−424. (31) Delaney, J. L.; Hogan, C. F.; Tian, J.; Shen, W. Anal. Chem. 2011, 83, 1300−1306. (32) Delaney, J. L.; Doeven, E. H.; Harsant, A. J.; Hogan, C. F. Anal. Chim. Acta 2013, 790, 56−60. (33) Lillehoj, P. B.; Huang, M.-C.; Truong, N.; Ho, C.-M. Lab Chip 2013, 13, 2950−2955. (34) Zhu, H.-Y.; Mavandadi, S.; Coskun, A. F.; Yaglidere, O.; Ozcan, A. Anal. Chem. 2011, 83, 6641−6647. (35) Martinez, A. W.; Phillips, S. T.; Carrilho, E.; Thomas, S. W.; Sindi, H.; Whitesides, G. M. Anal. Chem. 2008, 80, 3699−3707. (36) Mudanyali, O.; Dimitrov, S.; Sikora, U.; Padmanabhan, S.; Navruz, I.; Ozcan, A. Lab Chip 2012, 12, 2678−2686. (37) Chang, B.-Y. Bull. Korean Chem. Soc. 2012, 33, 549−552. (38) Oncescu, V.; O’Dell, D.; Erickson, D. Lab Chip 2013, 13, 3232− 3238. (39) Oncescu, V.; Mancuso, M.; Erickson, D. Lab Chip, DOI: 10.1039/c1033lc51194d.
2732
dx.doi.org/10.1021/ac404135f | Anal. Chem. 2014, 86, 2727−2732
