Simultaneous Electrochemical and Emission Monitoring of

Subscriber access provided by UNIV OF BARCELONA

Article

Simultaneous Electrochemical and Emission Monitoring of Electrogenerated Chemiluminescence through Instrument Hyphenation Andrew Scott Danis, Jesse B Gordon, Karlie Paige Potts, Lisa Irene Stephens, Samuel Charles Perry, and Janine Mauzeroll Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04960 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 8, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

Analytical Chemistry

Simultaneous Electrochemical and Emission Monitoring of Electrogenerated Chemiluminescence through Instrument Hyphenation Andrew S. Danis, Jesse B. Gordon, Karlie P. Potts, Lisa I. Stephens, Samuel C. Perry, Janine Mauzeroll* Department of Chemistry, McGill University, 801 Sherbrooke Street West, Montreal H3A 0B8, QC, Canada Email: [email protected] Fax Number: 514-398-3797

One of the long standing challenges to performing electrogenerated chemiluminescence (ECL) research is the need for dedicated instrumentation or highly customized cells to achieve reproducibility. This manuscript describes an approach to designing ECL systems through the hyphenation of existing laboratory instruments, which provide innate time correlation of electrochemical and emission data. This design methodology lowers the entry barrier required to obtaining reproducible ECL measurements, and provides flexibility in the scope of applications. Uniquely, the simplicity of this system’s experimental interface, a spectrochemical quartz cuvette, readily enables collaboration with finite element modeling that simulates ECL occurring in the cuvette based cell. This combination of empirical and simulation data allowed for the investigation of the intertwined kinetics behind the coreactant ECL mechanism of tris(2,2’-bipyridine)ruthenium(II) (Ru(bpy)32+) and tripropylamine (TPA). The complexity of the system measurable via the hyphenation methodology was further scaled though the addition of tris[2-(4,6-difluorophenyl)pyridinato-C2, N] iridium(III) (Ir(dFppy)3) and the observation of real time multiplexing.

INTRODUCTION Electrogenerated chemiluminescence (ECL) as a field has been a subject of interest for electrochemists since it was reported in the mid 1960’s.1 Its ability to be used in analytical assays would be recognized 20 years later.2 Today, ECL research thrives across a spectrum of fields.3 Its broad appeal is a consequence of researchers seeking to harness the intrinsic advantages that ECL provides over other light emission based techniques, which include a theoretical zero background, localized emission, and high selectivity.4,5 The challenge to employing this electrochemically generated excited state lies in developing instrumentation that can function in both the domains of electrochemistry and spectroscopy. Fundamentally, an ECL system must be able to observe light emission from electrogenerated intermediates at an electrode.4 This at a minimum requires the combination of electrochemical and chemiluminescent functionalities in an ECL system. A common trend in emission-centric ECL instrumentation is the optimization of the light collection capabilities to such an extent that electrochemistry is rendered a means to an end, instead of a highly interrogative tool. The ramification of this is electrochemical characterization of the electrochemically driven process is then performed separate from light emission collection.6–8 In contrast, an ECL system fabricated via the hyphenation of instruments facilitates the simultaneous employment of both analytical techniques in a single experiment.9 Consequently, the full investigative utility provided by both of these instruments is employed under the exact same experimental conditions. This not only reduces the overall experimental time and required number of experimental ECL instruments, but facilitates the direct temporal correlation of results.

Equally, if not more important than interfacing the instrument’s electronics, is interfacing the instruments experimentally. Routinely, laboratories develop a highly specialized experimental cell to research a specific ECL system or characteristic, this practice limits the accessibility of ECL research to those with identical resources.10 Instead, this work uses a spectroscopic cuvette as the electrochemical cell. Thus negating any difficulties associated with fabrication, cost, and integration of light collection optics. To validate this hyphenated system, the analytical benchmark for coreactant driven ECL, Ru(bpy)32+ and TPA,11 was examined through a range of concentrations. The data obtained by the hyphenated system was not limited to a calibration curve that defined the experimental boundaries of the system. Due to the cuvette based ECL cell’s well defined geometries, the ECL occurring in the hyphenated system can be computationally modeled.12 This feature transforms the hyphenated system into a powerful tool capable of pairing experimental and simulated results and enables novel insights into systems as complex as coreactant ECL. The role of this hyphenated system is not limited to fundamental investigations and can be extended through instrument selection or substitution. The hyphenated system described in this work utilized a Czerny Turner spectrometer as the photodetector. While trading the absolute sensitivity of a PMT, the increased spatial resolution of the spectrometer allowed the system to demonstrate multiplexing functionality. ECL based multiplexing in research has been achieved predominantly by two methodologies. One uses the applied potential to separate the observable signals over time.13 The other discriminates against multiple signals reporting simultaneously at different wavelengths.14 The capability to resolve the emission of multiple luminophores in real time

ACS Paragon Plus Environment

Analytical Chemistry 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

Page 2 of 8

facilitates development of analytically attractive multiplexing assays where multiple target analytes are tracked simultaneously.15–17 The inherent flexibility provided by being able to select and later substitute the instruments makes the hyphenated development of ECL systems, described herein, highly accessible to either laboratories looking to commence ECL research or to reduce the required amount of dedicated ECL instrumentation.

RESULTS AND DISCUSSION Instrument Coordination and Synchronization. The goal of simultaneously interrogating novel ECL platforms for both electrochemical and spectroscopic information created the demand for a system capable of reproducibly performing both measurements in sync. This manuscript serves as a practical guide for developing an ECL system through the hyphenation of native laboratory equipment, specifically, a potentiostat and spectrometer, without sacrificing either instrument’s functionality. Figure 1 displays the sequence of events performed by the hyphenated system to conduct an ECL experiment controlled using a single, central computer that simultaneously operated the software suites of both the potentiostat and spectrometer. Synchronization of the commercial potentiostat and spectrometer was achieved through utilization of a single analog trigger responsible for initiating both instruments simultaneously. The analog trigger was initiated by the potentiostat receiving a signal from the central computer. The trigger was then transmitted as a rising voltage step edge via a DB9 connection from the potentiostat to a trigger-in pin on a DB15 connection of the spectrometer. Once the trigger was received by the spectrometer, both instruments were synchronized in initiating their set experimental protocols and commenced recording and transmitting data to the central computer. The resulting temporal correlation of the electrochemistry and spectroscopy is demonstrated in Figure 2. The figure showcases the traces of current (monitored by the potentiostat) and ECL intensity (monitored by the spectrometer) over multiple cycles of cyclic voltammetry. Both the order of appearance and relative timing between the peaks through the course of experiment provides high confidence that the instruments are synchronized (See SI Synchronization and Troubleshooting). Considering the emission process of ECL is elicited by electrogenerated intermediates, a rise in current should proceed ECL emission, which is evident in Figure 2. The other corroborating indicator is the consistency in temporal spacing between the current and ECL peaks per cyclic voltammogram cycle. The temporal resolution of the hyphenated system is limited by the instrument with the lowest sampling frequency. In this setup, it is the spectrometer with a maximum integration time of approximately 10 Hz. This is further reduced by the signal averaging present in our data treatment methodology to the order of seconds.

Figure 1. ECL system coordination and synchronization. The following architecture was used to interface the individual instruments during an experiment: 1) The computer sends a signal activating the potentiostat. 2) The potentiostat sends an analog trigger activating the spectrometer. 3) The potentiostat begins applying programmed electrochemical waveforms at the ECL cuvette. Simultaneously, both the potentiostat and spectrometer measure the chemical system’s response to the applied potentials in the forms of current and light emission, respectively. Recorded data is transmitted back to the computer. 4) The spectrometer detects ECL emission once a threshold voltage is achieved to form reactive chemical intermediates.

Figure 2. Synchronization of potentiostat and spectrometer measurements. Current response (black) recorded by potentiostat at 1 ms sampling interval overlaid with simultaneous ECL response recorded (red) by spectrometer using 0.1 s integration time and integrating wavelengths 550 to 700 nm. The potential waveform utilized was a cyclic voltammogram with voltages of 0 V to 1.3 V to 0 V vs Ag/Ag+ and a scan rate of 100 mV/s for 6 cycles. The solution was 10 mM TPA, 1 mM Ru(bpy)32+, and 0.1 M TBAPF6 in acetonitrile.

2 ACS Paragon Plus Environment

Page 3 of 8 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

Analytical Chemistry Hyphenated System’s ECL Data Treatment. A CzernyTurner style spectrometer was selected as the light detecting element in the ECL system for its capability to balance temporal and spectral resolution. The drawback in using a high sensitivity instrument to detect a transient emission signal is that it is highly susceptible to random and systematic noise over the course of a measurement. This can reduce precision in measurements by either skewing the background or altering the intensity of the signal (See SI Data Treatment Methodology). A methodology was developed to treat the data from the spectrometer that focused on minimizing background noise and removing any outlier pixels. Intensity data was initially smoothed using a 10 point boxcar average; when compared to sampling at a lower frequency, this method better mitigates random noise while still providing adequate temporal resolution for the scan rate of 100 mV/s. This is because the signal to noise (S/N) ratio for a regime governed by random noise should increase with n1/2 post averaging, where n is the number of points averaged.18 After a background subtraction was performed to remove any slope in the measurement, a series of threshold criteria were applied to identify random pixels with significant deviation from the population average as outliers. The criteria were based on a pixel being three standard deviations away from mean intensity, mean range, or mean variance of the rest of the population. Figure 3 demonstrates the effect of the data treatment methodology on the spectrometer’s signal to noise ratio. The black trace represents the raw spectrometer data’s S/N profile at a set time point during ECL emission. The red trace represents the same data after the data treatment methodology. The luminophore’s emission observed in this experiment was Ru(bpy)32+, which characteristically emits between 550-700 nm.19 This band of emission provides an important positive control for the treatment methodology. That is, it should not artificially generate signal in regions where no emission is observed, which is evident in the non-emission region of the spectra covering 350-549 nm. In the region where the signal is observed, the developed methodology serves to markedly increase the S/N ratio, specifically near the emission maximum for Ru(bpy)32+ , 620 nm.20 Hyphenated System’s ECL Calibration Curve. A benefit of the hyphenated ECL system is being able to examine the electrochemical response in conjunction with spectral emission for a data set. After evaluating a series of concentrations, quantitative comparison of the emission data was limited to the first cycle of cyclic voltammetry. This selection provided the highest signal intensity, i.e. highest sensitivity, among the six cycles for both emission and current (See Figure 2). Chemically, this results from the maximum concentration of reagents available to the electrode occurring prior to the first cycle and being diminished upon every subsequent cycle. This decision also provided the highest precision across replicates as selecting only the first cycle prevented products from prior reactions in previous cycles from influencing ECL intensity. While the electrochemical data provides diagnostic and mechanistic information, focusing on the system’s ECL emission capabilities provided validation of the hyphenated system’s fidelity. This is found in the replication of wellestablished ECL trends for the luminophore/coreactant system,

Figure 3. Signal to noise comparison of ECL emission intensity per wavelength before (black) and after (red) data treatment. ECL emission obtained by spectrometer using a solution of 15 mM TPA, 25 μM Ru(bpy)32+, and 0.1 M TBAPF6 in MeCN. Original data was recorded by spectrometer using an integration rate of 100 ms. Data treatment used a 10 point boxcar and a series of filters to increase the signal to noise ratio of ECL emission. The background standard deviation was calculated from non-faradaic timeframes between 50-100 s, and the signal was taken from the 1st ECL peak value at 114.5 s.

Ru (bpy)32+/ TPA. In Figure 4A, an exponential relationship between coreactant, TPA concentration, and the resulting signal intensity is observed at relatively similar ratios of coreactant to luminophore. This relationship demonstrates the significant contribution to light emission that TPA plays in the ECL coreactant mechanism,21,22 as well as providing an efficient way to enhance emission intensity for ECL based detection systems. In Figure 4B, a linear relationship between ECL intensity and luminophore concentration at large excess of coreactant is observed. Consequently, these regimes of coreactant and luminophore provide not only enhanced ECL emission, but also regions of linearity that can be exploited for analytical assays.22– 24

COMSOL Model Simulating Experimental Trends. Despite a vast number of studies examining and utilizing this Ru(bpy)32+/ TPA system,17,25,26 the kinetics facilitating the complex mechanism remain largely speculative due to the intertwined nature of multiple competing pathways.27,28 Building off of a pre-established model for the cuvette based ECL cell,12 simulation was able to qualitatively match the experimentally generated calibration curves (see Figure 5). The previous iteration of the model examined the coreactant mechanism using spectroelectrochemistry and tracked concentration of the luminophore;29 this version of the model directly interrogated ECL emission from the coreactant mechanism by simulating the excited state species concentration. Under the accepted coreactant mechanism,28 the rate of heterogeneous TPA oxidation at the



Figure 4. Calibration curves of Ru(bpy)32+ TPA systems, generated from the integrated ECL intensity collected over wavelengths 550700 nm using the emission from the first cycle of cyclic voltammetry. Each concentration was repeated four times and error bars represent standard deviation. The electrochemistry protocol was a 100 s chronoamperogram at 0 V vs Ag/Ag+ followed by cyclic voltammetry with vertex potentials 0 V to 1.3 V to 0 V vs Ag/Ag+ at 100 mV/s for 6 cycles. A) Calibration curve of ECL emission with a varied TPA concentrations and fixed luminophore at 1 mM Ru(bpy)32+. B) Calibration curve for ECL emission using varied luminophore, Ru(bpy)32+, concentration and fixed coreactant, 15 mM TPA.

electrode had to be accelerated in this model to facilitate reproducing the nonlinear experimental trends (Figure 5A). Otherwise, linearity was strictly observed. The other caveat this version of the model identified when reproducing the experimental data was different relative kinetic rates among the

Page 4 of 8

competing homogeneous reactions. This model found the fastest homogeneous reaction in the mechanism was the electron transfer between the Ru(II) species and the deprotonated TPA radical. This could imply a kinetic control governing the fastest rate of electron transfer versus the thermodynamic control believed before.29 Alternatively, this could imply a non-uniform electron transfer excited state conversion efficiency between the competing reactions. While these results do not precisely reconcile with the previous findings,29 it maintains the importance of continued investigation using these types of lab built systems in combination with advanced modeling. Hyphenated System’s ECL Multiplexing. The functionality of the hyphenated system extends beyond mechanistic investigation of model systems. The selection of a spectrometer with high temporal resolution as the photon detector facilitates the system’s capability resolve multiple luminophores emitting simultaneously. To assess this functionality, a second luminophore, Ir(dFppy)3,13 which emits light at lower wavelengths than Ru(bpy)32+, was added to a solution of Ru(bpy)32+ and TPA. Since both of these luminophores undergo an oxidative-reductive coreactant mechanism independently with TPA,30 both should be able to form simultaneous excited states under the right concentration ratios (Figure 6A). Figure 6B shows the simultaneous emission bands of both luminophores over time. Ru(bpy)32+ and Ir(dFppy)3 occupy the bands from 550-700 nm and 450-550 nm respectively. The graphs in Figure 6C and 6D illustrate the versatility of the data that can be extracted from a single multiplexing experiment. Figure 6C shows the emission profiles for both emitters. The Ru(bpy)32+ control demonstrates significantly decreased emission intensity despite being at a matched concentration with the Ru(bpy)32+ in the multiplexing experiment. This is due to a known additional pathway where Ru(bpy)32+ in the presence of Ir luminophores will undergo electron transfer with the Ir luminophores producing Ru(bpy)32+* resulting in elevated emission intensities.31 Figure 6D displays the emission intensities for regions bounded by a single luminophore over time. This validates the hyphenated ECL system’s ability to track multiple luminophores emission in time. CONCLUSIONS The lab-built hyphenated ECL system presented herein provides researchers access to a wide range of ECL applications. The fundamental hardware required to construct these versatile hyphenated systems is merely a potentiostat, photodetector, cuvette, and computer. Besides the single point of control for both instruments, hyphenation adds value by synchronizing the actions of separate instruments, which facilitates the direct time correlation of empirical results. This feature, in combination with the established geometries of a cuvette based ECL cell, made it possible to provide the simulation framework necessary to develop the only coreactant ECL model currently investigating the kinetics of the Ru(bpy)32+/TPA mechanism. The inherent flexibility in the instruments employable enables these types of systems to perform advanced ECL research, which was demonstrated by real-time multiplexing of iridium and ruthenium luminophores. Despite the elementary nature of these hyphenation-based setups, the increased obtainability of reproducible ECL data


Page 5 of 8 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

Analytical Chemistry provides both the current ECL community and those looking to enter a valuable tool for the way ahead.

Figure 5. Overlay of simulated ECL intensity with experimental observed ECL Intensity. Comparison of the simulated ECL emission intensity (blue) and experimentally observed ECL emission intensity (black) for different ratios of coreactant and luminophore. Simulated ECL intensities were determined through

integrating the concentration of exited state species Ru(bpy)32+*over time. The electrochemical protocol employed in the model was cyclic voltammetry with vertex potentials of 0.3 V, 1.3 V, and 0.3 V vs Ag/Ag+ A) Simulation with varied TPA concentrations and fixed luminophore at 1 mM Ru(bpy)32+. B) Simulation with varied luminophore, Ru(bpy)32+, concentration and fixed coreactant, 15 mM TPA.

EXPERIMENTAL SECTION Materials. Experiments were conducted utilizing tris(2,2’bipyridine)ruthenium(II) hexafluorophosphate (Ru(bpy)32+) (Sigma-Aldrich), tetrabutylammonium hexafluorophosphate (TBAPF6) (Sigma Aldrich), tripropylamine (TPA)(Basf), tris[2-(4,6-difluorophenyl)pyridinato-C2, N] iridium(III) (Ir(dFppy)3) (Sigma-Aldrich), HPLC grade acetonitrile (MeCN) (Fisher), dichloromethane (DCM) (ACP), chloroplatinic acid (Sigma-Aldrich), ethanol (EtOH) (Commercial Alcohols), Acetone (ACP), and distilled H2O. Drying agents used were magnesium sulfate (MgSO4) (SigmaAldrich) and molecular sieves (ACP). Epoxies utilized in electrode construction were a two part conductive Ag epoxy (EPO-TEK) and the three part reinforcing DuroCIT-3 epoxy (Struers) Equipment. The potentiostat and spectrometer utilized in the design of the ECL instrument were a VSP300 (Biologic) and Maya LSL (Ocean Optics). The light collection optics consisted of a ∅ 10 mm aspherical condenser lens (ACL108) (Thorlabs) and a 1 m fiber optic (Ocean Optics) directing emitted light to the spectrometer. The electrochemical cell was constructed inside a quartz cuvette (Hellma Analytics). Polymer shims securing electrodes at the bottom of the cuvette were made from Versaflex CL30 (GLS Corp). The cuvette cap was a custom 3D printed piece of Z-PETG using the Zortax M200. The electrochemistry was performed in a three electrode setup using a platinized piece of machined FTO (MSE Supplies LSE) for a working electrode, Pt mesh counter electrode (Good-fellow), and commercial Ag/Ag+ (10 mM Ag+ + e-→Ag) reference electrode Instrument Coordination and Synchronization. The timeline for instrument interfacing, applying stimuli, and recording data followed the same sequence for each experiment. At time zero a start signal was sent to the potentiostat from the computer. The potentiostat then performed two actions in sequence: it first sent a start trigger to the spectrometer, then began applying the voltage waveform dictated by the user. Simultaneous sampling of the photon emission and electrochemical current were performed by the spectrometer and potentiostat respectively, which transmitted the collected data back to the central computer for storage, display, and analysis.



Page 6 of 8

Figure 6. Multiplexing capability of spectrometer based cuvette system. A) An example of dual simultaneous emission mechanism for Ru(bpy)32+ and Ir(dFppy)3 utilizing a common electrogenerated coreactant specie. The solution utilized was 40 uM Ru(bpy)32+ and 5 mM Ir(dFppy)3 and 10 mM TPA. B) Spectrometer data post treatment demonstrating versatility of analysis for collected data through examining spectra along Y-axis (wavelength) to generate figure C or along X-axis (time) to generate figure D. C) Intensity vs wavelength applied at 12.5 s displaying two emission profiles for Ru(bpy)32+ and Ir(dFppy)3. D) Intensity vs time collected over resolved regimes allowing isolation of Ru(bpy)32+ versus Ir(dFppy)3 emission.

ECL Experiment Preparation. All glassware (with the exception of the cuvette) was first cleaned in an ethanoate bath overnight, then rinsed with distilled water, EtOH, and Acetone. After drying in air, glassware was placed in 120 °C oven overnight. Prior to solution preparation, all solvents were placed in round bottom flasks and either dried via molecular sieves or chemically with MgSO4. Assembly of Electrochemical Cuvette. Prior to loading the ECL cuvette with a sample aliquot, the quartz cuvette, shims, and electrodes were rinsed with EtOH, acetone, and air dried. The cuvette, shims, and electrodes were then rinsed with dried MeCN followed by rinsing with several hundred μL’s of sample solution. The polymer shims were positioned at the bottom of the cuvette to orient and reproducibly stabilize the electrodes

position (See SI ECL Cuvette Design). When used in combination with the electrode cap at the top of the cuvette, the working electrode would be centered and the counter electrode would be fixed at the rear of the cuvette. The cuvette was then assembled through fitting the working and counter electrodes in the cuvette cap and guiding the bottoms of the electrodes into their respective positions (See SI ECL Cuvette Design). A 2.5 mL aliquot of sample solution was finally loaded to fill the cuvette before the reference electrode was inserted. Hyphenated System’s ECL Data Treatment. After an ECL experiment was performed, it was necessary to manually treat the data from the spectrometer. The output generated by the proprietary software often contained slopes making baseline integration of peaks challenging. To combat this, raw data


Page 7 of 8 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

Analytical Chemistry exported from the spectrometer was first refined to the spectral region of interest. A boxcar average was then performed to smooth the intensity recorded by each pixel over time. A line of best fit was generated for each pixel’s smoothed intensity over the course of an experiment. The value for this line of best fit was then subtracted from each pixel to remove any slope throughout the measurement. At low signal to noise levels, ECL peaks could be obscured by several individual pixels that had significantly deviated in intensity recording from the majority pixels. Three criteria were applied to identify and remove outlier pixels based on parameters derived from intensity. These criteria included isolating pixels demonstrating significant deviations from the mean pixel intensity, mean pixel range, and mean pixel variance over the timeframe of the experiment. The wavelengths associated with these pixels were random with each experiment and the removed pixels do not exceed 5% of the total amount (See SI Data Treatment Methodology). An automated ECL peak emission integration methodology was developed to assess each ECL peak. It required first establishing the noise value for each experiment by integrating the last 50 s of a chronoamperogram. The chronoamperogram was set to a potential that elicited zero faradaic response for the coreactant system, 0 V vs Ag/Ag+. An initial estimate for determining the limits of integration for ECL emission was established by setting a wide fixed boundary around the time point corresponding to the current max value for each cycle of cyclic voltammetry. Inside this boundary, intensity was integrated at one second intervals to compare what intervals, if any, were above the noise value. The ECL integration boundaries were then refined to start when the first interval’s intensity value increased above noise, and end when an interval’s intensity decreased back to noise. Total ECL intensity per cycle was determined through numerical integration of these refined boundaries using a trapezoid methodology (See SI Data Treatment Methodology). Hyphenated System’s ECL Calibration Curve. After the trigger from the potentiostat to the spectrometer, the first electrochemical waveform applied was a chronoamperogram holding a potential of 0 V vs Ag/Ag+ for 100 s to allow the system to stabilize. This was followed by 6 cyclic voltammograms with vertex potentials of 0 V to 1.3 V vs Ag/Ag+ and used a scan rate of 100 mV/s. The spectrometer’s sampling frequency was 10 Hz, and emission was monitored in the region from 550-700 nm. The values for ECL intensity per cycle reported were calculated via the data processing methodology described in hyphenated system’s ECL data treatment. Ru(bpy)32+ and TPA samples were analyzed starting from the least concentrated and progressing to the most concentrated solution for the specific species of interest. Each experiment was performed with 4 replicates. Between each replicate the ECL cuvette was deconstructed, washed, rinsed, and reassembled as described in the electrochemical cuvette assembly procedures. COMSOL Model Simulating ECL Trends. A 2D numerical model was built in COMSOL Multiphysics 5.3a as described previously.12,29 Heterogeneous kinetics at the electrode were described according to the Butler-Volmer equation, and homogeneous kinetics in solution were described according to traditional reaction order kinetics. The full model

details may be found in the supporting information (See SI Simulation Methodology). Hyphenated System’s ECL Multiplexing. The electrochemical protocol for the multiplexing experiment was identical to that used in the calibration curve. The spectrometers sampling frequency was 10 Hz, and collected ECL emission wavelengths from 350-700 nm. A 5 mM solution of Ir(dFppy)3, 10 mM TPA ,and 0.1 M TBAPF6 in MeCN:DCM (80:20) was prepared. An aliquot was extracted and analyzed as the Ir(dFppy)3 control. A separate 40 M Ru(bpy)32+, 10 mM TPA, and 0.1 M TBAPF6 MeCN solution was utilized as the Ru(bpy)32+ control. To the remaining Ir(dFppy)3/TPA solution an aliquot from a 1 mM Ru(bpy)32+ 0.1 M TBAPF6 MeCN solution was added to make the Ru(bpy)32+ in the multiluminophore solution approximately 40 M. The sample solution was then analyzed in the same manner as the control.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publication website. Contained within the SI is: ECL Cuvette Design, Synchronization Troubleshooting, Electrode Optimization, Data Treatment Methodology, and Simulation Methodology

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel: 514-398-3898

Present Addresses Jesse B. Gordon: Department of Chemistry, MIT, 77 Massachusetts Avenue, 18-393, Cambridge, MA 02139 Samuel C. Perry: Faculty of Engineering and Physical Sciences, University of Southampton, University Road, Southampton SO17 1BJ

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Funding Sources J.M. was supported by a team operational grant from Fonds de Recherche Nature et Technologies Quebec (FRQNT) (No. PR175718)

ACKNOWLEDGMENT We would like to acknowledge Dr Sylvain Canesi and Dr. Hanadi Sleiman for their support.

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8)

Hercules, D. M. Science (80). 1964, 145 (3634), 808–809. Ege, D.; Becker, W. G.; Bard, A. J. Anal. Chem. 1984, 56 (13), 2413–2417. Miomandre, F.; Audebert, P. Luminescence in electrochemistry: Applications in analytical chemistry, physics and biology; 2016. Miao, W. Chem. Rev. 2008, 108 (7), 2506–2553. Benoit, L.; Choi, J. P. ChemElectroChem 2017, 4 (7), 1573–1586. Qi, H. et al. J. Am. Chem. Soc. 2016, 138 (6), 1947–1954. MSD. Meso Scale Discovery ECL Instruments https://www.mesoscale.com/en/products_and_services/instrume ntation (accessed Oct 16, 2018). Block Scientific, I. Roche ELECSYS 2010 Immunoassay



(9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)

Analyzer https://www.blockscientificstore.com/Roche-Elecsys2010-p/roche-elecsys-2010.htm (accessed Oct 16, 2018). Hirschfeld, T. Anal. Chem. 1980, 52 (2), 297A–312A. Nepomnyashchii, A. B.; Cho, S.; Rossky, P. J.; Bard, A. J. J. Am. Chem. Soc. 2010, 132 (49), 17550–17559. Pyati, R.; Richter, M. M. Annu. Rep. Prog. Chem. Sect. C Phys. Chem. 2007, 103 (0), 12–78. Danis, A. S. et al. ChemElectroChem 2017, 4 (7), 1736–1743. Kerr, E. et al. Chem. Sci. 2015, 6 (1), 472–479. Muegge, B. D.; Richter, M. M. Anal. Chem. 2004, 76 (1), 73–77. Zhou, J.; Nie, L.; Zhang, B.; Zou, G. Anal. Chem. 2018. Volpe, A.; Kurtys, E.; Fruhwirth, G. O. Int. J. Biochem. Cell Biol. 2018, 102, 40–50. Kadimisetty, K. et al. Biosens. Bioelectron. 2016, 77, 188–193. Ingle, J. D.; Crouch, S. R. Spectrochemical Analysis; Prentice Hall: Upper Saddle River, 1988. McCord, P.; Bard, A. J. J. Electroanal. Chem. Interfacial Electrochem. 1991, 318 (1), 91–99. Knight, A. W.; Greenway, G. M. Analyst 1996, 121 (11), 101R– 106R.

(21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31)

Page 8 of 8 Zu, Y.; Bard, A. J. Anal. Chem. 2000, 72 (14), 3223–3232. Sun, S. et al. Anal. Chem. 2009, 81 (24), 10227–10231. Guo, Z. et al. Analyst 2018, 143 (14), 3353–3359. Lu, H. J. et al. Anal. Chem. 2018, 90 (17), 10434–10441. Sardesai, N. P.; Barron, J. C.; Rusling, J. F. Anal. Chem. 2011, 83 (17), 6698–6703. Wang, T.; Wang, D.; Padelford, J. W.; Jiang, J.; Wang, G. J. Am. Chem. Soc. 2016, 138 (20), 6380–6383. Miao, W.; Choi, J. P.; Bard, A. J. J. Am. Chem. Soc. 2002, 124 (48), 14478–14485. Svir, I.; Oleinick, A.; Klymenko, O. V.; Amatore, C. ChemElectroChem 2015, 2 (6), 811–818. Danis, A. S.; Potts, K. P.; Perry, S. C.; Mauzeroll, J. Anal. Chem. 2018. Liu, Z.; Qi, W.; Xu, G. Chem. Soc. Rev. 2015, 44 (10), 3117– 3142. Kerr, E. et al. Chem. Sci. 2016, 7 (8), 5271–5279.

Table of Contents (TOC) Figure:


Simultaneous Electrochemical and Emission Monitoring of

Recommend Documents