- Email: [email protected]
Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrene-graphite composite electrodes
Analytica Chimica Acta xxx (2017) 1e7
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrene-graphite composite electrodes €ivi Gro € nroos a, Sami Tuomi b, Sakari Kulmala a Kalle Salminen a, *, Pa a b
Laboratory of Analytical Chemistry, Department of Chemistry, Aalto University, P.O. Box 16100, 00076 Aalto, Finland Research Group of Electrochemical Energy Conversion and Storage, Department of Chemistry, Aalto University, P.O. Box 16100, 00076 Aalto, Finland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Generation of hydrated electrons at Polystyrene-graphite electrodes. The insulating polystyrene layer on the outer electrode surface seems necessary. Hydrated electrons are able to produce chemiluminescence. Strongest signal and lowest std. dev. achieved at same graphite weight fraction.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2016 Received in revised form 4 July 2017 Accepted 12 July 2017 Available online xxx
Tb(III) chelates exhibit intense hot electron-induced electrogenerated chemiluminescence during cathodic polarization of metal/polystyrene-graphite (M/PG) electrodes in fully aqueous solutions. The M/ PG working electrode provides a sensitive means for the determination of aromatic Tb(III) chelates at nanomolar concentration levels with a linear log-log calibration curve spanning more than five orders of magnitude. The charge transport and other properties of these novel electrodes were studied by electrochemiluminescence measurements and cyclic voltammetry. The present composite electrodes can by utilized both under pulse polarization and DC polarization unlike oxide-coated metal electrodes which do not tolerate cathodic DC polarization. The present cost-effective electrodes could be utilized e.g. in immunoassays where polystyrene is extensively used as a solid phase for various bioaffinity assays by using electrochemiluminescent Tb(III) chelates or e.g. Ru(bpy)2þ 3 as labels. © 2017 Elsevier B.V. All rights reserved.
Keywords: Hot electron electrochemistry Electrochemiluminescence Lanthanide luminescence Bioaffinity assays
1. Introduction The hydrated electron can be seen as the smallest anion and the simplest reactive chemical species existing in aqueous chemistry. They are commonly encountered in e.g. pulse radiolysis studies, sonolysis studies, glow discharge studies, flash photolysis and even when pieces of alkali metals are dissolved into water [1e6]. One way of utilizing the strong reducing power and fast reaction rates of
* Corresponding author. E-mail address: kalle.salminen@aalto.fi (K. Salminen).
hydrated electrons is in the generation of hot electron-induced electrochemiluminescence (HECL). HECL is an method in analytical chemistry in which hydrated electrons generated by electrical pulses are used to mediate chemical reactions in solution between otherwise nonreactive chemical species [7]. These externally triggered redox reactions can lead to the formation of excited states that can subsequently relax to the ground state by emitting electromagnetic radiation. HECL has a considerable potential in various fields of Point-of-Care and Point-of-Need analysis e.g. in immunoassays or other bioassays by the virtue of high sensitivity and inexpensive instrumentation. The working electrode (cathode) is typically some kind of conducting material (metal or a degenerate
http://dx.doi.org/10.1016/j.aca.2017.07.035 0003-2670/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
2
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
highly doped semiconductor) uniformly covered with a thin insulating film with a thickness of less than ten nanometers. Frequently used working electrode materials include Al/Al2O3, Mg/MgO, Si/ SiO2 and n-ZnO:Al/MgO with oxide-coated aluminum being the most common due to the naturally forming Al2O3-layer [8e10]. The insulating film has the following properties: 1) it allows the electrode to act as a tunnel emitter/cold cathode during cathodic pulse polarization 2) it restricts ordinary cathodic reactions that occur below the onset electric field of the tunnel emission process 3) it hinders the return of emitted electrons back to the metal and 4) it prevents the metal/conductor from quenching the excited states of the luminophore [11]. Polystyrene is an organic insulating polymer commonly used in disposable plastic products where rigid and low-cost plastic is required. It is the most commonly used material among solid phase macro carriers (e.g. micro titer strips, balls and tubes) used in heterogeneous immunoassays [12]. In HECL, the insulating polystyrene film on the cathode surface can likewise function as a binding material for biomolecules under study making such electrodes easily adoptable to various bioassays using existing coating methods for different biomolecules. For example DELFIA® assays and many other bioassays are routinely carried out on the polystyrene surface. Polystyrene beads have previously been used e.g. in immunoassays for the determination of C-reactive protein and human thyroid stimulating hormone and these analytes have also been previously studied in our laboratory with HECL [13e16]. Other useful properties of polystyrene include good chemical resistivity, wide usable pH-range and optical transparency. Thin polymer films of uniform thickness can be manufactured in a laboratory setting by e.g. spin coating, dip-coating or drop casting. For example Chen et al. reported using paraffin oil covered cathode electrodes in HECL that provided satisfactory results for the determination of catechol [17]. Insulating organic polymer film covered electrodes are however not well suited for commercial or practical laboratory use due to sluggish manufacturing speeds, strict quality control requirements, fragile nature of the polymer layer and the need for expensive and clean substrates. One solution to these issues, that makes printable polymerbased electrodes a viable replacement for typical metal/metal oxide-electrodes, is by using composite electrodes made of insulating polymer and suitable conducting particles as presented in this work. In the present article the performance of metal/ polystyrene-graphite (M/PG) electrodes in HECL was studied in detail as a potential low-cost replacements for expensive oxidecoated silicon electrodes [15]. 2. Experimental Polystyrene (Mw ¼ 350.000, Sigma-Aldrich) was dissolved overnight in analytical grade chloroform at 50 g L1 unless otherwise mentioned. Graphite flakes (4e10 mm in diameter, Forward Working Group, England) were added at various weight ratios and the solutions were mixed at 13 000 RPM (T-10 homogenizer, IKA) for one hour to ensure homogenous, aggregate-free dispersion. Freshly dispersed suspensions were spin coated to 0.05 mm thick brass substrates (15 mm in diameter). Brass was selected as the conducting substrate for polystyrene-graphite electrodes, since it itself is incapable of producing HECL from any luminophore. Prior to spin coating the substrates were cleaned in ultrasonic bath (70 W) with acetone. Cleaned substrates were allowed to dry overnight. Spin coating deposition was performed in normal room atmosphere at 3500 RPM for 60 s. The sheet resistivity of composite electrodes were measured with Jandel RM3000 test unit equipped with a cylindrical four point probe head capable of measuring sheet resistivities up to 500 MU ,1. For resistivity measurements the
ink was spin coated to soda lime glass with same conditions as above. Electrode surface was characterized by cyclic voltammetry with a typical three-electrode setup. For this experiment the ink was spin coated to glassy carbon electrode with the same conditions as above. The HECL measurements were made in cylinder Teflon cell containing an integrated vertical platinum wire counter electrode that was positioned in the middle of the cell. Distance between electrodes was approximately 1 mm. Excitation of luminophores was done with either an in-laboratory-built coulostatic pulse generator delivering constant charge voltage pulses (35 V, charge 12.6 mC, frequency 20 Hz, 2000 pulses) or with a simple DC voltage supply (20 V, 60 mA). HECL measurements were carried out with an electrochemiluminometer composed of Nucleus MCS-II multiscaler card, photomultiplier tube module (PMT) and Stanford Research SR400 Photon Counter. With pulse polarization excitation the luminescence was measured with time-resolved (TR) technique. The light was measured for four milliseconds after the end of each excitation pulse. A 545 nm interference filter with 40 nm bandwidth was used to pass only the strongest spectral line from Tb(III) to the PMT. The M/PG electrodes were used once and discarded thereafter. Terbium(III) 4-(Phenyl-ethyl)(1-hydroxybenzene)-[2,6pyridinediylbis(methylene nitrilo)]tetrakis(acetic acid) was obtained from University of Turku. The model luminophore was dissolved in freshly made 0.05 M borate buffer (pH ¼ 9.2) that has very low reactivity towards hydrated electrons and other radicals [18]. Two other aromatic Tb(III) chelates with phenolic moieties were also shortly tested and all of them showed strong electrochemiluminescence at the present composite electrodes. K2S2O8, Na2B4O7$10H2O, NaNO3 and NaNO2 were pro analysis grade reagents from Merck. Long exposure time photographs (15 s) of HECL were taken in completely dark room with Canon EOS 7D digital camera equipped with Canon EF 100 mm f/2.8 L Macro IS USM lens. The camera and coulostatic pulse generator were turned on simultaneously. All photos had the brightness increased by the same amount to make the emission more visible for the reader. HECL spectra was measured with Andor Technology DV465C-FI electron multiplying charge-coupled device (EMCCD) detector attached to an Oriel Instruments MS125 (model 77400) spectrograph. 3. Results and discussion 3.1. HECL at metal/polystyrene-graphite electrodes Various metal/polystyrene-graphite (M/PG) composite electrodes were used as the cathode electrodes in the electrochemiluminescence of Tb(III) chelate/peroxydisulfate -system. Long exposure-time photographs (Fig. 1) were taken during the cathodic pulse polarization excitation with the camera pointing straight down to the circular electrochemical cell, the Pt-wire counter electrode blocked some of the generated emission due to positioning. The characteristic green emission of terbium was observed at the planar cathode electrode when the weight fraction of graphite (wg) was roughly between five and fifty percent. Both observed intensity per pixel and the amount of green pixels in the photographs was highly dependent on the wg value. When the graphite concentration was increased from five to thirty percent the observed emission intensity increased substantially. This is most likely due to increase in the amount of graphite flakes that are capable of generating hydrated electrons, decrease in the average distance between outermost graphite flakes and the electrolyte solution and increase in the PG-composite conductivity. Further increase in wg caused the observed emission to disappear almost completely.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
3
Fig. 1. Long exposure photographs of 103 M Tb(III) chelate, 103 M S2O28 -system.
3.2. Primary cathodic processes of HECL
3.3. The effect of free radical scavengers on HECL Assuming that the previously investigated excitation pathways are valid in the present study, hot or hydrated electron are the primary reducing species and, if no co-reactants (e.g. S2O2 8 ) are present, hydroxyl radicals generated from the dissolved oxygen are the primary efficiently oxidizing species. Consequently, free-radical scavengers are expected to have a significant effect on the observed emission intensity. Nitrate (Fig. 2 triangles) and nitrite (Fig. 2 circles) ions are rapid hydrated electron scavengers with secondorder reaction rate constants of 9.77$109 mol1 s1 and 4.17$109 mol1 s1 respectively [18]. The observed quenching effects are in agreement with the reaction rate constants, suggesting that the M/PG electrodes are capable of generating hydrated electrons and highlighting the importance of hydrated electrons in the light generation pathways as discussed previously. The observed emission intensity in HECL can be enhanced by the addition of co-reactants that produce one-electron oxidants upon one-electron reduction. The dependency of the TR-HECL signal on the concentration of K2S2O8 (Fig. 2 squares) is similar to previous studies done with different electrode materials such as Al/Al2O3 and Si/Si2O3 [24,25]. The ability of SO4 to react mainly as oneelectron oxidant in comparison to OH- leads to almost hundredfold increase in the observed emission intensity when the concentration of K2S2O8 reaches 103 M. However, when the concentration of K2S2O8 is increased beyond this value the observed HECL intensity starts to decrease due to the imbalance between reducing and oxidizing species i.e. there is too little amount of hydrated
TR-HECL (photon counts)
The primary cathodic process with insulating film-coated electrodes has been shown to be a tunnel emission/field-assisted direct tunneling of hot electrons (e hot) into aqueous electrolyte solution [19]. When these electrons are injected into the conduction band of water they can became hydrated in femtosecond time-scale (e aq) and mediate highly energetic chemical reactions that are not feasible with traditional electrochemistry at active metal electrodes. The light forming pathway for the Tb(III) chelate/S2O2 8 -system is based on redox-reaction pathways discussed in greater detail elsewhere [8]. In addition to the insulating film-covered electrodes discussed previously there are studies on the field emission of electrons into liquid medium from electrochemically active electrodes such as carbon nanotubes, Pt-, W-, carbon and Cuwires [20e23]. The literature on this topic suggests that the tip of the electrode has to be extremely sharp for the electron field emission to occur at reasonable cathodic voltages [22]. Our research group recently reported on HECL of Ru(bpy)2þ 3 at sharpened Pt-wire electrode during cathodic pulse polarization and it is possible that similar phenomena could occur in the present study due to miniature field emission points provided by the sharp graphite flakes protruding from polystyrene matrix [23]. It.
107
106
105
104
103 10-6
10-5
10-4 10-3 10-2 Concentration (mol L-1)
10-1
Fig. 2. Effect of electron scavengers: NO 3 (triangles), NO2 (circles) and co-reactant K2S2O8 (squares) on the TR-HECL intensity in 105 M Tb(III) chelate system. Wg ¼ 30%.
electrons available to reduce the Tb(III) chelate or its one-electron oxidized form [24]. Taken together with the scavenger measurements it is highly probable that hydrated electrons are involved in the excitation process in the present case and that the visible luminescence from terbium chelate showed in Fig. 1 is not due to a heterogeneous electron transfer at the cathode surface or by energy transfer from the solid state electroluminescence centers.
3.4. The effect of graphite content and charge transport through the PG-layer The graphite weight fraction range at which M/PG electrodes are capable of exciting the Tb(III) chelate was investigated further (Fig. 3 hollow squares). When pure polystyrene (50 g L1) was spin coated to brass substrate, no light emission from Tb(III) chelate was observed. Increase in the excitation voltage led to dielectric breakdown of the PS film. Therefore, electron injection into the electrolyte solution with M/PG electrodes has to occur exclusively from the graphite particles, not from the brass substrate underneath the PG composite itself. The composite electrodes were capable of producing varying amounts of luminescence from the aromatic terbium chelate when the wg was between one and sixty-five percent. The emitting species was confirmed by measuring the emission spectrum and calculating the luminescence lifetime of the Tb(III) chelate. When wg was increased from 1% to 10% the emission intensity increased tenfold and stayed relatively same until 30% after which the emission intensity started to decline.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
4
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
Fig. 3. Sheet resistivity of electrodes (hollow diamonds), TR-HECL (hollow squares) and coefficient of variation (black circles). 105 M Tb(III) chelate, 103 M K2S2O8.
The highest usable wg value was approximately 40%. In addition to producing only a fraction of TR-HECL intensity than the electrodes with lower wg values, the reproducibility of the TR-HECL signal between electrodes was substantially worse beyond this graphite concentration (Fig. 3 black circles). After wg was increased beyond 55% some of the electrodes failed to produce any Tb(III) chelate specific luminescence at all with increasing wg value correlating with higher electrode failure rate. All of the electrodes with less than 60% wg were capable of generating HECL, even those that had just one percent graphite. The failure to generate HECL is assumable caused by the highly irregular introduction of bare graphite flakes on electrode/electrolyte-interface which starts to interfere with the tunneling process and lowers the observed TRHECL intensity which ultimately prevents the electrons from tunneling altogether when wg> 65%. Similar behavior regarding the relationship between the insulation layer coverage and measured HECL-intensity has been reported previously with MgO- and paraffin/graphite-electrodes [10,17]. Electrodes with Wg values higher than 53% were incapable of exciting the Tb(III) chelate at the beginning of the experiment. However, when the pulse polarization was continued the emission
from Tb(III) chelate was suddenly observed at gradually increasing intensity (Fig. 4 curves b and c), the amount of preconditioning pulses needed for the generation of HECL increased with the Wg value. This odd behavior was not seen with electrodes containing lesser amounts of graphite (Fig. 4 curve a). It is likely that a passivation of bare graphite particles by hydrogen bubbles or some other kind of surface passivation takes place that effectively blocks the “leaking” charge transport from the active graphite rich electrode areas on the surface. This charge “leaking” otherwise prevents the charge transfer to solution via electron tunneling i.e. this leaking current from active carbon particles on the surface to the solution species can only produce hydrogen evolution but not HECL. After electrodes were washed, dried and reused they behaved similarly as in the first measurement. This supports the idea of uninsulated graphite particles being the cause for nonfunctioning working electrodes and hydrogen bubbles being the source of passivation process that makes HECL generation possible after some induction time during pulse-polarization. While electron emission occurs exclusively from the outermost graphite particles, it is noteworthy to consider the charge transport mechanism through the polystyrene layer. The measured sheet resistance (Fig. 3, hollow diamonds) as a function of wg forms a “S”shaped curve that is composed of three different regions, however only two parts of the curve are visible due to instruments inability to measure values above the MU-range. These different regions each correspond to different dominant charge transport mechanism inside the composite (hopping, tunneling-percolation and coalescence), that are in turn related to the average distance between adjacent particles [26]. The first percolation threshold (wg ¼ approx. 25% in the case of M/PG electrodes) for conductivity is observed when the inter-particle distance between conducting particles is ten nanometers and results in global electrical conductivity within the composite [27]. M/PG electrodes produced HECL (i.e. they were able to generate hydrated electrons) in every charge transport mechanism region. The highest emission intensity, and the lowest standard deviation, was observed around the offset of coalescence network that occurs at approximately wg ¼ 30%. The sheet resistance at this value is unfortunately rather large at 100 kU,1 and the conducting particles should be switched to e.g. conductive carbon black or carbon nanotubes if polystyrene-carbon-composite electrodes were to be made without a conducting substrate underneath the composite film itself or if the composite layer is made thicker. Electrodes with low graphite concentrations managed produced HECL even though their resistivity matches closely that of pure polystyrene: 1015 U ,1 [28]. These electrodes were however plagued with lower HECL emission intensity and much higher variation in the emission intensity. The latter is caused by the low probability of outermost graphite particles being “electrically connected” to other graphite flakes/brass substrate as no global conducting networks exists at low carbon loading values. It seems reasonable to assume that for similar composite materials, especially those having the similar graphite particles, the optimal concentration of conducting particles is located near the coalescence offset point. 3.5. Durability of the disposable electrodes
Fig. 4. TR-HECL luminogram for M/PG electrodes: wg ¼ 50, 60 and 65% for a, b and c respectively. 105 M Tb(III) chelate, 103 M K2S2O8.
To study the durability of composite electrodes the HECL was measured for 4000 excitation cycles, which is ten times more than is typically used for HECL immunoassays [15]. Stable emission intensity was observed during the whole measurement, indicating that the electrode had good electrochemical stability (Fig. 5 curve a). To investigate the effect of acids and bases on the electrode performance they were immersed either in 3 M sodium hydroxide (curve b) or concentrated sulfuric acid (curves c-e) for various
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
Fig. 5. Effect on acid/base treatment on emission intensity with M/PG electrodes (wg ¼ 30%). No pretreatment (a), 10 min in 3 M sodium hydroxide (b), 30 s, 5 and 10 min in sulfuric acid (c,d and e respectively). 106 M Tb(III) chelate, 103 M K2S2O8.
times. The treatment with the former seemed to have only a meager effect on HECL-emission intensity. When electrodes were pretreated with concentrated sulfuric acid the change in emission intensity was more drastic, more than a hundredfold decrease in emission intensity was observed when the electrodes were soaked in sulfuric acid for ten minutes before the HECL measurement. This can be explained by the partial removal of the polystyrene layer, which is supported by the fact that increase in acid treatment time seemed to increase the number of pulses it took to obtain highest emission intensity due to more unexposed graphite particles present on the electrode surface. The observed decrease in the emission intensity with both sodium hydroxide and sulfuric acid pretreatments gives further validation for the importance of the insulating layer with the present composite electrodes. 3.6. M/PG electrodes under DC polarization When direct current (DC) is used to drive the electron tunneling process with aluminum electrodes the insulating layer is disintegrated within milliseconds when voltage is applied to the electric circuit [29]. Quite similarly, when DC excitation was used with the M/PG electrodes the light emission was observed only for a couple of seconds before a sudden crash in the emission intensity (Fig. 6, curve b), after which the emission intensity abruptly increased and stayed at the same level for rest of the measurement. The steep decrease in emission intensity is caused by a mechanical failure of the PG composite that was observed to be detached from the brass substrate. However, a thin layer of PG composite still has to exist on the brass surface that makes the hot electron emission possible. The violent detachment of the composite is likely caused by the generation of hydrogen gas/bubbles in the vicinity of the composite/brass interface, which was not observed with pulse polarization due to less charge being transported and the periodical nature of charge injection. Thicker PG electrodes were spin coated which helped to alleviate this problem completely (Fig. 6 curves a and c). The steady decrease in emission intensity during measurement is observed both for Tb(III) chelate/S2O2 8 -system and S2O28 -blank and therefore likely related to consumption of peroxide which is known to increase the blank emission in HECL [30]. Another ill effect caused by the vigorous gas formation during the DC excitation is the noisier nature of the emission intensity signal, which coupled with the inability to perform time resolved
5
Fig. 6. DC excitation, electrodes (wg ¼ 30%) made from inks containing 50 (b) or 200 (a,c) g L1 polystyrene. 104 M Tb(III) chelate, 103 M K2S2O8 (curves a,b) and blank (c).
measurements makes the detection limits considerably worse. On the other hand, the use of DC excitation simplifies the instrumentation even further. 3.7. M/PG electrodes as studied by cyclic voltammetry Cyclic voltammetry (CV) was used to analyze the surface of the glassy carbon/PG electrodes (Fig. 7), i.e. to determine if the electrodes with varied composition would show active or passive behavior. If one electron reduction of ferricyanide to ferrocyanide takes place on the graphite surface the measured peak current is governed by Randles-Sevcik relationship which states that peak current is linearly dependent on electroactive surface-area. In order for electron transfer to occur, the energy levels of the donor states within the carbon and the acceptor levels within the solution must be in resonance. The voltammogram for wg ¼ 30% electrode shows that the surface of the M/PG composite is passivated, i.e. graphite particles are not in direct contact with the electrolyte solution, which makes the recorded faradaic current non-existent. As shown
Fig. 7. CV for various electrodes. Uncoated glassy carbon (solid line), glassy carbon electrode coated with graphite/polystyrene mixture containing 30% (dashed line), 50% (dotted line) and 55% (dot dashed line) of graphite. 100 mV s1 scan rate in 1 mM [Fe(CN)6]4 solution with 1 M KCl as the supporting electrolyte.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
6
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
previously in Fig. 1B the light forming chemical reactions are taking place practically near the whole cathode surface. Area this large should have been effortlessly detectable by CV if it was not covered by polystyrene layer, as the average distance it takes electron to localize as well as the average distance they drift-diffuse before reacting with water is estimated to be 2.5 ± 1.0 nm [31]. At 55% graphite concentration a clear oxidation peak was observed at 0.3 V (vs. SCE), but the charge transfer kinetics are very slow as seen from the high DEp-value, suggesting that a degree of electrical insulation around the graphite flakes exists even at higher graphite flake values. The different electrochemical response of electrodes shown in Fig. 7 complements results shown in Figs. 1 and 3. Bare graphite flakes are undoubtedly present on the electrolyte interface after M/PG electrodes contain more than 53% of graphite and the initial mechanism of charge transport during cathodic pulse polarization is normal electrochemical reduction taking place directly on the cathode surface. It is likely that bare graphite flakes are present at the electrode/electrolyte interface even at lower graphite concentrations as the observed emission intensity started to decline after Wg reached values beyond 30%. However, if this is the case, the amount of bare graphite has to be small as the CV measurements lacked the oxidation peak for electrodes containing 50% of graphite. In the light all these measurements it seems that the electron injection from M/PG electrodes is based on electron tunnel emission trough insulating layer rather than some kind of field emission from bare graphite flakes. 3.8. Analytical applicability of M/PG electrodes Log-log calibration curve for the blank-corrected Tb(III) chelate/ S2O2 8 -system is shown in Fig. 8 for both DC (squares) and pulse polarization excitation (circles). Good correlation between Tb(III) chelate concentration and measured TR-HECL signal was obtained in both cases, with pulsed excitation the error bars at higher chelate concentrations are smaller than the data points themselves. The detection limit (s/n ¼ 3, rounded up to the nearest power of ten) was 109 M for pulse polarization and 106 M with DC excitation. Presented in the inset of Fig. 8 are the decay profiles for blank and nanomolar Tb(III) chelate emissions. The amount of excitation
Fig. 8. Blank corrected calibration curve of Tb(III) chelate/103 M S2O2 8 -system with pulsed- (filled circles) or DC- excitation (empty squares), wg ¼ 30 %for both cases, n ¼ 7 for each data point. Inset: decay of emission intensity, blank (b) and 109 M Tb(III) chelate (a).
pulses was triple the amount used in the standard curve measurements to make the difference between the blank and sample emissions easier to observe visually. The difference in the luminescence lifetimes allows for effortless distinction between these two signals when HECL is measured by time-resolved technique. In HECL the following factors are affected by the working electrode: the amount of hydrated electrons generated (i.e. intensity of TR-HECL signal), intrinsic electroluminescence from the electrode material (noise that affects the s/n-value) and the reproducibility of TR-HECL signal between electrodes. Repeatability was examined by measuring seven disposable electrodes. The standard deviation for the measured Tb(III) chelate emission at 105 M concentration was 4.3% when the excitation was done with pulse polarization and roughly 23% when DC excitation was used. This shows that the performance of M/PG electrodes is sufficiently good to warrant further research. 3.9. Blank emission under cathodic polarization Electroluminescence (EL) is the main contributor to blank signal in HECL. With thin insulating film-coated electrodes the EL is mainly based on impact excitation of luminescent impurities and on electron center luminescence (F-center, E-center, etc.) at the insulator/electrolyte interface [32]. The lowest background emission is obtained when the oxide film does not contain any luminescent impurities and encloses as low number of defect states as possible. Because the polystyrene can be mixed with variety of different conducting particles, each having a unique EL fingerprint, the EL was measured for pure polystyrene thin-films and for M/PG composite electrodes (Fig. 9). The pure polystyrene films were spin coated from solution containing only 5 g L1 polystyrene. With these metal/polystyrene-electrodes the short-lived electroluminescence appears only during the cathodic potential pulse and fades away instantly when the excitation pulse ends (Fig. 9 curves c and d). For M/PG electrodes (wg ¼ 30%) there is clearly long-lived luminescence of some origin (Fig. 9 curves a and b) that lowers the s/n-ratio considerably, even when optical filtration is used and when the luminophore has a long luminescence lifetime. The purity of graphite flake powder (99.53%) was measured with X-ray fluorescence analysis, impurities included silicon, phosphorous, aluminum, sulfur and less than 0.05% of Ca, Mg, Cu and Pb.
Fig. 9. Decay of electroluminescence in borate buffer solution. Plotted with respect to the end of the excitation pulse. M/PG electrode (wg ¼ 30%) without (a) and with optical filter (b). Pure polystyrene thin film without (c) and with a filter (d). 4000 excitation pulses.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
4. Conclusions Hot electron-induced electrochemiluminescence of Tb(III) chelate/S2O28 -system in aqueous solutions at metal/polystyrenegraphite composite electrodes was investigated for the first time. The excitation of the aromatic Tb(III) chelate was mainly based on the injection of hot electrons into electrolyte solution from the graphite particles embedded in the polystyrene matrix. Due to the generation of solvated electrons the metal/polystyrene-graphite composite electrodes are capable of exciting other luminophores such as Ru(bpy)2þ 3 as well [33]. The observed emission intensity was profoundly dependent on the amount of graphite particles present in/on the composite material. The graphite weight fraction range at which the electrodes were capable of exciting the Tb(III) chelate was very wide: 1e65%. The insulating polystyrene layer at the electrolyte interface seemed to be necessary for the efficient generation of hydrated electrons and subsequent electrochemiluminescence. Detection limit for Tb(III) chelate was ca. 109 M with pulse- and 106 M with DC-excitation. When more suitable conducting particles than presently used are applied, polystyrene based composite electrodes can be suggested to fully replace silicon disc electrodes in low-cost disposable plastic cartridges, making it reasonable to use silicon electrodes only in those cases when sophisticated electrode chips are used and fabricated in sufficiently large batches [15,34]. Funding Preliminary experiments of these studies were supported by SalWe Oy/Technology development center of Finland (534/14). References [1] M.J. Bronskill, Picosecond pulse radiolysis studies. I. The solvated electron in aqueous and alcohol solutions, J. Chem. Phys. 53 (1970) 4201e4210, http:// dx.doi.org/10.1063/1.1673922. [2] P. Cintas, J.-L. Luche, Green chemistry. A sonochemical approach, Green Chem. 1 (1999) 115e125, http://dx.doi.org/10.1039/a900593e. [3] J. Goodman, A. Hickling, B. Schofield, The yield of hydrated electrons in glowdischarge electrolysis, J. Electroanal. Chem. Interfacial Electrochem 48 (1973) 319e322, http://dx.doi.org/10.1016/S0022-0728(73)80272-4. [4] F. Tochikubo, Y. Shimokawa, N. Shirai, S. Uchida, Chemical reactions in liquid induced by atmospheric-pressure dc glow discharge in contact with liquid, Jpn. J. Appl. Phys. 53 (2014) 126201, http://dx.doi.org/10.7567/JJAP.53.126201. [5] M.S. Matheson, W.A. Mulac, J. Rabani, formation of the hydrated electron in the flash photolysis of aqueous solutions 1, J. Phys. Chem. 67 (1963) 2613e2617, http://dx.doi.org/10.1021/j100806a027. k, T. Buttersack, S. Bauerecker, P. Jungwirth, [6] P.E. Mason, F. Uhlig, V. Vane Coulomb explosion during the early stages of the reaction of alkali metals with water, Nat. Chem. 7 (2015) 250e254, http://dx.doi.org/10.1038/ nchem.2161. [7] S. Kulmala, J. Suomi, Current status of modern analytical luminescence methods, Anal. Chim. Acta 500 (2003) 21e69, http://dx.doi.org/10.1016/ j.aca.2003.09.004. [8] S. Kulmala, A. Kulmala, T. Ala-Kleme, J. Pihlaja, Primary cathodic steps of electrogenerated chemiluminescence of lanthanide(III) chelates at oxidecovered aluminum electrodes in aqueous solution, Anal. Chim. Acta 367 (1998) 17e31, http://dx.doi.org/10.1016/S0003-2670(98)00154-8. [9] S. Kulmala, T. Ala-Kleme, L. Heikkil€ a, L. V€ are, Energetic electrochemiluminescence of (9-fluorenyl)methanol induced by injection of hot electrons into aqueous electrolyte solution, J. Chem. Soc. Faraday Trans. 93 (1997) 3107e3113, http://dx.doi.org/10.1039/a702135f. [10] M. Håkansson, Q. Jiang, M. Helin, M. Putkonen, A.J. Niskanen, S. Pahlberg, T. Ala-Kleme, L. Heikkil€ a, J. Suomi, S. Kulmala, Cathodic Tb(III) chelate electrochemiluminescence at oxide-covered magnesium and n-ZnO: Al/MgO composite electrodes, Electrochim. Acta 51 (2005) 289e296, http://dx.doi.org/ 10.1016/j.electacta.2005.04.033. [11] T. Ala-Kleme, S. Kulmala, Q. Jiang, Generation of free radicals and electrochemiluminescence from simple aromatic molecules in aqueous solutions, Luminescence 21 (2006) 118e125, http://dx.doi.org/10.1002/bio.895. [12] V.V. Shmanai, T.A. Nikolayeva, L.G. Vinokurova, A.A. Litoshka, Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(meth)acrylic acid, BMC Biotechnol. 1 (2001) 4, http://dx.doi.org/10.1186/1472-6750-1-4.
7
[13] E. Ishikawa, Y. Hamaguchi, M. Imagawa, M. Inada, H. Imura, N. Nakazawa, H.O. Daiichi, An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay, J. Immunoass. 1 (1980) 385e398, http:// dx.doi.org/10.1080/01971528008058479. [14] S. Kulmala, M. Håkansson, A.M. Spehar, A. Nyman, J. Kankare, K. Loikas, T. AlaKleme, J. Eskola, Heterogeneous and homogeneous electrochemiluminoimmunoassays of hTSH at disposable oxide-covered aluminum electrodes, Anal. Chim. Acta 458 (2002) 271e280, http://dx.doi.org/10.1016/ S0003-2670(02)00065-X. [15] T. Ylinen-Hinkka, A.J. Niskanen, S. Franssila, S. Kulmala, Immunoassay of Creactive protein by hot electron induced electrochemiluminescence using integrated electrodes with hydrophobic sample confinement, Anal. Chim. Acta 702 (2011) 45e49, http://dx.doi.org/10.1016/j.aca.2011.06.038. [16] J.O. Koskinen, J. Vaarno, N.J. Meltola, J.T. Soini, P.E. H€ anninen, J. Luotola, M.E. Waris, A.E. Soini, Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology, Anal. Biochem. 328 (2004) 210e218, http://dx.doi.org/10.1016/ j.ab.2004.02.029. [17] X.Y. Chen, R.J. Zheng, S.F. Qin, J.J. Sun, Hot electron-induced cathodic electrochemiluminescence at oil film-covered carbon paste electrode and application to nano-molar determination of catechol, Talanta 101 (2012) 362e367, http://dx.doi.org/10.1016/j.talanta.2012.09.042. [18] G.V. Buxton, C.L. Greenstock, W.P. Helman, A.B. Ross, Critical Review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (,OH/,O) in Aqueous Solution, J. Phys. Chem. Ref. Data 17 (1988) 513e886, http://dx.doi.org/10.1063/1.555805. [19] S. Suomi, J. Kulmala, Reviews in Fluorescence 2009, Springer-Verlag, New York, 2009, http://dx.doi.org/10.1007/s10895-005-2528-0. [20] A.G. Krivenko, N.S. Komarova, E.V. Stenina, L.N. Sviridova, V.A. Kurmaz, A.S. Kotkin, V.E. Muradyan, Electrochemical behavior of electrodes containing nanostructured carbon of various morphology in the cathodic region of potentials, Russ. J. Electrochem 42 (2006) 1047e1054, http://dx.doi.org/10.1134/ S1023193506100090. [21] A.G. Krivenko, N.S. Komarova, Electron injection from nanostructured carbon electrodes at moderate cathodic potentials, Russ. J. Electrochem 43 (2007) 1123e1126, http://dx.doi.org/10.1134/S1023193507100035. [22] A.G. Krivenko, N.S. Komarova, P.N.P, Electrochemical generation of solvated electrons from nanostructured carbon, Electrochem. Commun. 9 (2007) 2364e2369, http://dx.doi.org/10.1016/j.elecom.2007.07.004. [23] P. Kuosmanen, K. Salminen, M. Pusa, T. Ala-Kleme, S. Kulmala, Hot electroninduced electrogenerated chemiluminescence of Ru(bpy)2þ chelate at a 3 pointed active metal cathode in fully aqueous solutions, J. Electroanal. Chem. 738 (2016) 63e67, http://dx.doi.org/10.1016/j.jelechem.2016.10.057. [24] T. Ylinen, J. Suomi, M. Helin, T. Ala-Kleme, S. Kulmala, Time-resolved detection of hot electron-induced electrochemiluminescence of fluorescein in aqueous solution, J. Fluoresc. 16 (2006) 27e33, http://dx.doi.org/10.1007/s10895-0050023-2. [25] S. Kulmala, K. Haapakka, Mechanism of electrogenerated luminescence of terbium(III)-“2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-4benzoylphenol” chelate at an oxide-covered aluminium electrode, J. Alloy. Compd. 225 (1995) 502e506, http://dx.doi.org/10.1016/0925-8388(94) 07058-X. [26] S. Nakamura, K. Saito, G. Sawa, K. Kitagawa, Percolation threshold of carbon black-polyethylene composites, Jpn. J. Appl. Phys. Part 1 Regul. Pap. Short. Notes Rev. Pap. 36 (1997) 5163e5168, http://dx.doi.org/10.1143/JJAP.36.5163. [27] J. Li, J.K. Kim, Percolation threshold of conducting polymer composites containing 3D randomly distributed graphite nanoplatelets, Compos. Sci. Technol. 67 (2007) 2114e2120, http://dx.doi.org/10.1016/j.compscitech.2006.11.010. [28] M. Salovey, R. Ghofraniha, Electrical conductivity of polymers containing carbon black, Polym. Eng. Sci. 28 (1988) 58e63, http://dx.doi.org/10.1002/ pen.760280110. [29] M. Håkansson, Q. Jiang, A.M. Spehar, J. Suomi, M. Kotiranta, S. Kulmala, Direct current-induced electrogenerated chemiluminescence of hydrated and chelated Tb(III) at aluminum cathodes, Anal. Chim. Acta 541 (2005) 171e177, http://dx.doi.org/10.1016/j.aca.2004.12.072. [30] S. Kulmala, T. Ala-Kleme, A. Hakanen, K. Haapakka, F-Centre luminescence from oxide-covered aluminium cathode induced by two-step reduction of peroxydisulfate anions, J. Chem. Soc. Faraday Trans. 93 (1997) 165e168, http://dx.doi.org/10.1039/a604025j. [31] P. Rumbach, D.M. Bartels, R.M. Sankaran, D.B. Go, The solvation of electrons by an atmospheric-pressure plasma, Nat. Commun. 6 (2015) 7248, http:// dx.doi.org/10.1038/ncomms8248. [32] M. Håkansson, Q. Jiang, J. Suomi, K. Loikas, M. Nauma, T. Ala-Kleme, J. Kankare, P. Juhala, J.U. Eskola, S. Kulmala, Cathodic electrochemiluminescence at double barrier Al/Al2O3/Al/Al2O3 tunnel emission electrodes, Anal. Chim. Acta 556 (2006) 450e454, http://dx.doi.org/10.1016/j.aca.2005.09.064. [33] T. Ala-Kleme, S. Kulmala, L. Vare, P. Juhala, M. Helin, Hot electron-induced electrogenerated chemiluminescence of Ru(bpy)2þ 3 chelate at oxide covered aluminum electrodes, Anal. Chem. 71 (1999) 5538e5543, http://dx.doi.org/ 10.1021/ac981336i. [34] M. Håkansson, K. Salminen, P. Kuosmanen, J. Eskola, H. Peuravuori, S. Kulmala, Immunoassay of b2-microglobulin at oxide-coated heavily doped p-silicon electrodes, J. Electroanal. Chem. 769 (2016) 11e15, http://dx.doi.org/10.1016/ j.jelechem.2016.02.040.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrene-graphite composite electrodes €ivi Gro € nroos a, Sami Tuomi b, Sakari Kulmala a Kalle Salminen a, *, Pa a b
Laboratory of Analytical Chemistry, Department of Chemistry, Aalto University, P.O. Box 16100, 00076 Aalto, Finland Research Group of Electrochemical Energy Conversion and Storage, Department of Chemistry, Aalto University, P.O. Box 16100, 00076 Aalto, Finland
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Generation of hydrated electrons at Polystyrene-graphite electrodes. The insulating polystyrene layer on the outer electrode surface seems necessary. Hydrated electrons are able to produce chemiluminescence. Strongest signal and lowest std. dev. achieved at same graphite weight fraction.
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 November 2016 Received in revised form 4 July 2017 Accepted 12 July 2017 Available online xxx
Tb(III) chelates exhibit intense hot electron-induced electrogenerated chemiluminescence during cathodic polarization of metal/polystyrene-graphite (M/PG) electrodes in fully aqueous solutions. The M/ PG working electrode provides a sensitive means for the determination of aromatic Tb(III) chelates at nanomolar concentration levels with a linear log-log calibration curve spanning more than five orders of magnitude. The charge transport and other properties of these novel electrodes were studied by electrochemiluminescence measurements and cyclic voltammetry. The present composite electrodes can by utilized both under pulse polarization and DC polarization unlike oxide-coated metal electrodes which do not tolerate cathodic DC polarization. The present cost-effective electrodes could be utilized e.g. in immunoassays where polystyrene is extensively used as a solid phase for various bioaffinity assays by using electrochemiluminescent Tb(III) chelates or e.g. Ru(bpy)2þ 3 as labels. © 2017 Elsevier B.V. All rights reserved.
Keywords: Hot electron electrochemistry Electrochemiluminescence Lanthanide luminescence Bioaffinity assays
1. Introduction The hydrated electron can be seen as the smallest anion and the simplest reactive chemical species existing in aqueous chemistry. They are commonly encountered in e.g. pulse radiolysis studies, sonolysis studies, glow discharge studies, flash photolysis and even when pieces of alkali metals are dissolved into water [1e6]. One way of utilizing the strong reducing power and fast reaction rates of
* Corresponding author. E-mail address: kalle.salminen@aalto.fi (K. Salminen).
hydrated electrons is in the generation of hot electron-induced electrochemiluminescence (HECL). HECL is an method in analytical chemistry in which hydrated electrons generated by electrical pulses are used to mediate chemical reactions in solution between otherwise nonreactive chemical species [7]. These externally triggered redox reactions can lead to the formation of excited states that can subsequently relax to the ground state by emitting electromagnetic radiation. HECL has a considerable potential in various fields of Point-of-Care and Point-of-Need analysis e.g. in immunoassays or other bioassays by the virtue of high sensitivity and inexpensive instrumentation. The working electrode (cathode) is typically some kind of conducting material (metal or a degenerate
http://dx.doi.org/10.1016/j.aca.2017.07.035 0003-2670/© 2017 Elsevier B.V. All rights reserved.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
2
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
highly doped semiconductor) uniformly covered with a thin insulating film with a thickness of less than ten nanometers. Frequently used working electrode materials include Al/Al2O3, Mg/MgO, Si/ SiO2 and n-ZnO:Al/MgO with oxide-coated aluminum being the most common due to the naturally forming Al2O3-layer [8e10]. The insulating film has the following properties: 1) it allows the electrode to act as a tunnel emitter/cold cathode during cathodic pulse polarization 2) it restricts ordinary cathodic reactions that occur below the onset electric field of the tunnel emission process 3) it hinders the return of emitted electrons back to the metal and 4) it prevents the metal/conductor from quenching the excited states of the luminophore [11]. Polystyrene is an organic insulating polymer commonly used in disposable plastic products where rigid and low-cost plastic is required. It is the most commonly used material among solid phase macro carriers (e.g. micro titer strips, balls and tubes) used in heterogeneous immunoassays [12]. In HECL, the insulating polystyrene film on the cathode surface can likewise function as a binding material for biomolecules under study making such electrodes easily adoptable to various bioassays using existing coating methods for different biomolecules. For example DELFIA® assays and many other bioassays are routinely carried out on the polystyrene surface. Polystyrene beads have previously been used e.g. in immunoassays for the determination of C-reactive protein and human thyroid stimulating hormone and these analytes have also been previously studied in our laboratory with HECL [13e16]. Other useful properties of polystyrene include good chemical resistivity, wide usable pH-range and optical transparency. Thin polymer films of uniform thickness can be manufactured in a laboratory setting by e.g. spin coating, dip-coating or drop casting. For example Chen et al. reported using paraffin oil covered cathode electrodes in HECL that provided satisfactory results for the determination of catechol [17]. Insulating organic polymer film covered electrodes are however not well suited for commercial or practical laboratory use due to sluggish manufacturing speeds, strict quality control requirements, fragile nature of the polymer layer and the need for expensive and clean substrates. One solution to these issues, that makes printable polymerbased electrodes a viable replacement for typical metal/metal oxide-electrodes, is by using composite electrodes made of insulating polymer and suitable conducting particles as presented in this work. In the present article the performance of metal/ polystyrene-graphite (M/PG) electrodes in HECL was studied in detail as a potential low-cost replacements for expensive oxidecoated silicon electrodes [15]. 2. Experimental Polystyrene (Mw ¼ 350.000, Sigma-Aldrich) was dissolved overnight in analytical grade chloroform at 50 g L1 unless otherwise mentioned. Graphite flakes (4e10 mm in diameter, Forward Working Group, England) were added at various weight ratios and the solutions were mixed at 13 000 RPM (T-10 homogenizer, IKA) for one hour to ensure homogenous, aggregate-free dispersion. Freshly dispersed suspensions were spin coated to 0.05 mm thick brass substrates (15 mm in diameter). Brass was selected as the conducting substrate for polystyrene-graphite electrodes, since it itself is incapable of producing HECL from any luminophore. Prior to spin coating the substrates were cleaned in ultrasonic bath (70 W) with acetone. Cleaned substrates were allowed to dry overnight. Spin coating deposition was performed in normal room atmosphere at 3500 RPM for 60 s. The sheet resistivity of composite electrodes were measured with Jandel RM3000 test unit equipped with a cylindrical four point probe head capable of measuring sheet resistivities up to 500 MU ,1. For resistivity measurements the
ink was spin coated to soda lime glass with same conditions as above. Electrode surface was characterized by cyclic voltammetry with a typical three-electrode setup. For this experiment the ink was spin coated to glassy carbon electrode with the same conditions as above. The HECL measurements were made in cylinder Teflon cell containing an integrated vertical platinum wire counter electrode that was positioned in the middle of the cell. Distance between electrodes was approximately 1 mm. Excitation of luminophores was done with either an in-laboratory-built coulostatic pulse generator delivering constant charge voltage pulses (35 V, charge 12.6 mC, frequency 20 Hz, 2000 pulses) or with a simple DC voltage supply (20 V, 60 mA). HECL measurements were carried out with an electrochemiluminometer composed of Nucleus MCS-II multiscaler card, photomultiplier tube module (PMT) and Stanford Research SR400 Photon Counter. With pulse polarization excitation the luminescence was measured with time-resolved (TR) technique. The light was measured for four milliseconds after the end of each excitation pulse. A 545 nm interference filter with 40 nm bandwidth was used to pass only the strongest spectral line from Tb(III) to the PMT. The M/PG electrodes were used once and discarded thereafter. Terbium(III) 4-(Phenyl-ethyl)(1-hydroxybenzene)-[2,6pyridinediylbis(methylene nitrilo)]tetrakis(acetic acid) was obtained from University of Turku. The model luminophore was dissolved in freshly made 0.05 M borate buffer (pH ¼ 9.2) that has very low reactivity towards hydrated electrons and other radicals [18]. Two other aromatic Tb(III) chelates with phenolic moieties were also shortly tested and all of them showed strong electrochemiluminescence at the present composite electrodes. K2S2O8, Na2B4O7$10H2O, NaNO3 and NaNO2 were pro analysis grade reagents from Merck. Long exposure time photographs (15 s) of HECL were taken in completely dark room with Canon EOS 7D digital camera equipped with Canon EF 100 mm f/2.8 L Macro IS USM lens. The camera and coulostatic pulse generator were turned on simultaneously. All photos had the brightness increased by the same amount to make the emission more visible for the reader. HECL spectra was measured with Andor Technology DV465C-FI electron multiplying charge-coupled device (EMCCD) detector attached to an Oriel Instruments MS125 (model 77400) spectrograph. 3. Results and discussion 3.1. HECL at metal/polystyrene-graphite electrodes Various metal/polystyrene-graphite (M/PG) composite electrodes were used as the cathode electrodes in the electrochemiluminescence of Tb(III) chelate/peroxydisulfate -system. Long exposure-time photographs (Fig. 1) were taken during the cathodic pulse polarization excitation with the camera pointing straight down to the circular electrochemical cell, the Pt-wire counter electrode blocked some of the generated emission due to positioning. The characteristic green emission of terbium was observed at the planar cathode electrode when the weight fraction of graphite (wg) was roughly between five and fifty percent. Both observed intensity per pixel and the amount of green pixels in the photographs was highly dependent on the wg value. When the graphite concentration was increased from five to thirty percent the observed emission intensity increased substantially. This is most likely due to increase in the amount of graphite flakes that are capable of generating hydrated electrons, decrease in the average distance between outermost graphite flakes and the electrolyte solution and increase in the PG-composite conductivity. Further increase in wg caused the observed emission to disappear almost completely.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
3
Fig. 1. Long exposure photographs of 103 M Tb(III) chelate, 103 M S2O28 -system.
3.2. Primary cathodic processes of HECL
3.3. The effect of free radical scavengers on HECL Assuming that the previously investigated excitation pathways are valid in the present study, hot or hydrated electron are the primary reducing species and, if no co-reactants (e.g. S2O2 8 ) are present, hydroxyl radicals generated from the dissolved oxygen are the primary efficiently oxidizing species. Consequently, free-radical scavengers are expected to have a significant effect on the observed emission intensity. Nitrate (Fig. 2 triangles) and nitrite (Fig. 2 circles) ions are rapid hydrated electron scavengers with secondorder reaction rate constants of 9.77$109 mol1 s1 and 4.17$109 mol1 s1 respectively [18]. The observed quenching effects are in agreement with the reaction rate constants, suggesting that the M/PG electrodes are capable of generating hydrated electrons and highlighting the importance of hydrated electrons in the light generation pathways as discussed previously. The observed emission intensity in HECL can be enhanced by the addition of co-reactants that produce one-electron oxidants upon one-electron reduction. The dependency of the TR-HECL signal on the concentration of K2S2O8 (Fig. 2 squares) is similar to previous studies done with different electrode materials such as Al/Al2O3 and Si/Si2O3 [24,25]. The ability of SO4 to react mainly as oneelectron oxidant in comparison to OH- leads to almost hundredfold increase in the observed emission intensity when the concentration of K2S2O8 reaches 103 M. However, when the concentration of K2S2O8 is increased beyond this value the observed HECL intensity starts to decrease due to the imbalance between reducing and oxidizing species i.e. there is too little amount of hydrated
TR-HECL (photon counts)
The primary cathodic process with insulating film-coated electrodes has been shown to be a tunnel emission/field-assisted direct tunneling of hot electrons (e hot) into aqueous electrolyte solution [19]. When these electrons are injected into the conduction band of water they can became hydrated in femtosecond time-scale (e aq) and mediate highly energetic chemical reactions that are not feasible with traditional electrochemistry at active metal electrodes. The light forming pathway for the Tb(III) chelate/S2O2 8 -system is based on redox-reaction pathways discussed in greater detail elsewhere [8]. In addition to the insulating film-covered electrodes discussed previously there are studies on the field emission of electrons into liquid medium from electrochemically active electrodes such as carbon nanotubes, Pt-, W-, carbon and Cuwires [20e23]. The literature on this topic suggests that the tip of the electrode has to be extremely sharp for the electron field emission to occur at reasonable cathodic voltages [22]. Our research group recently reported on HECL of Ru(bpy)2þ 3 at sharpened Pt-wire electrode during cathodic pulse polarization and it is possible that similar phenomena could occur in the present study due to miniature field emission points provided by the sharp graphite flakes protruding from polystyrene matrix [23]. It.
107
106
105
104
103 10-6
10-5
10-4 10-3 10-2 Concentration (mol L-1)
10-1
Fig. 2. Effect of electron scavengers: NO 3 (triangles), NO2 (circles) and co-reactant K2S2O8 (squares) on the TR-HECL intensity in 105 M Tb(III) chelate system. Wg ¼ 30%.
electrons available to reduce the Tb(III) chelate or its one-electron oxidized form [24]. Taken together with the scavenger measurements it is highly probable that hydrated electrons are involved in the excitation process in the present case and that the visible luminescence from terbium chelate showed in Fig. 1 is not due to a heterogeneous electron transfer at the cathode surface or by energy transfer from the solid state electroluminescence centers.
3.4. The effect of graphite content and charge transport through the PG-layer The graphite weight fraction range at which M/PG electrodes are capable of exciting the Tb(III) chelate was investigated further (Fig. 3 hollow squares). When pure polystyrene (50 g L1) was spin coated to brass substrate, no light emission from Tb(III) chelate was observed. Increase in the excitation voltage led to dielectric breakdown of the PS film. Therefore, electron injection into the electrolyte solution with M/PG electrodes has to occur exclusively from the graphite particles, not from the brass substrate underneath the PG composite itself. The composite electrodes were capable of producing varying amounts of luminescence from the aromatic terbium chelate when the wg was between one and sixty-five percent. The emitting species was confirmed by measuring the emission spectrum and calculating the luminescence lifetime of the Tb(III) chelate. When wg was increased from 1% to 10% the emission intensity increased tenfold and stayed relatively same until 30% after which the emission intensity started to decline.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
4
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
Fig. 3. Sheet resistivity of electrodes (hollow diamonds), TR-HECL (hollow squares) and coefficient of variation (black circles). 105 M Tb(III) chelate, 103 M K2S2O8.
The highest usable wg value was approximately 40%. In addition to producing only a fraction of TR-HECL intensity than the electrodes with lower wg values, the reproducibility of the TR-HECL signal between electrodes was substantially worse beyond this graphite concentration (Fig. 3 black circles). After wg was increased beyond 55% some of the electrodes failed to produce any Tb(III) chelate specific luminescence at all with increasing wg value correlating with higher electrode failure rate. All of the electrodes with less than 60% wg were capable of generating HECL, even those that had just one percent graphite. The failure to generate HECL is assumable caused by the highly irregular introduction of bare graphite flakes on electrode/electrolyte-interface which starts to interfere with the tunneling process and lowers the observed TRHECL intensity which ultimately prevents the electrons from tunneling altogether when wg> 65%. Similar behavior regarding the relationship between the insulation layer coverage and measured HECL-intensity has been reported previously with MgO- and paraffin/graphite-electrodes [10,17]. Electrodes with Wg values higher than 53% were incapable of exciting the Tb(III) chelate at the beginning of the experiment. However, when the pulse polarization was continued the emission
from Tb(III) chelate was suddenly observed at gradually increasing intensity (Fig. 4 curves b and c), the amount of preconditioning pulses needed for the generation of HECL increased with the Wg value. This odd behavior was not seen with electrodes containing lesser amounts of graphite (Fig. 4 curve a). It is likely that a passivation of bare graphite particles by hydrogen bubbles or some other kind of surface passivation takes place that effectively blocks the “leaking” charge transport from the active graphite rich electrode areas on the surface. This charge “leaking” otherwise prevents the charge transfer to solution via electron tunneling i.e. this leaking current from active carbon particles on the surface to the solution species can only produce hydrogen evolution but not HECL. After electrodes were washed, dried and reused they behaved similarly as in the first measurement. This supports the idea of uninsulated graphite particles being the cause for nonfunctioning working electrodes and hydrogen bubbles being the source of passivation process that makes HECL generation possible after some induction time during pulse-polarization. While electron emission occurs exclusively from the outermost graphite particles, it is noteworthy to consider the charge transport mechanism through the polystyrene layer. The measured sheet resistance (Fig. 3, hollow diamonds) as a function of wg forms a “S”shaped curve that is composed of three different regions, however only two parts of the curve are visible due to instruments inability to measure values above the MU-range. These different regions each correspond to different dominant charge transport mechanism inside the composite (hopping, tunneling-percolation and coalescence), that are in turn related to the average distance between adjacent particles [26]. The first percolation threshold (wg ¼ approx. 25% in the case of M/PG electrodes) for conductivity is observed when the inter-particle distance between conducting particles is ten nanometers and results in global electrical conductivity within the composite [27]. M/PG electrodes produced HECL (i.e. they were able to generate hydrated electrons) in every charge transport mechanism region. The highest emission intensity, and the lowest standard deviation, was observed around the offset of coalescence network that occurs at approximately wg ¼ 30%. The sheet resistance at this value is unfortunately rather large at 100 kU,1 and the conducting particles should be switched to e.g. conductive carbon black or carbon nanotubes if polystyrene-carbon-composite electrodes were to be made without a conducting substrate underneath the composite film itself or if the composite layer is made thicker. Electrodes with low graphite concentrations managed produced HECL even though their resistivity matches closely that of pure polystyrene: 1015 U ,1 [28]. These electrodes were however plagued with lower HECL emission intensity and much higher variation in the emission intensity. The latter is caused by the low probability of outermost graphite particles being “electrically connected” to other graphite flakes/brass substrate as no global conducting networks exists at low carbon loading values. It seems reasonable to assume that for similar composite materials, especially those having the similar graphite particles, the optimal concentration of conducting particles is located near the coalescence offset point. 3.5. Durability of the disposable electrodes
Fig. 4. TR-HECL luminogram for M/PG electrodes: wg ¼ 50, 60 and 65% for a, b and c respectively. 105 M Tb(III) chelate, 103 M K2S2O8.
To study the durability of composite electrodes the HECL was measured for 4000 excitation cycles, which is ten times more than is typically used for HECL immunoassays [15]. Stable emission intensity was observed during the whole measurement, indicating that the electrode had good electrochemical stability (Fig. 5 curve a). To investigate the effect of acids and bases on the electrode performance they were immersed either in 3 M sodium hydroxide (curve b) or concentrated sulfuric acid (curves c-e) for various
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
Fig. 5. Effect on acid/base treatment on emission intensity with M/PG electrodes (wg ¼ 30%). No pretreatment (a), 10 min in 3 M sodium hydroxide (b), 30 s, 5 and 10 min in sulfuric acid (c,d and e respectively). 106 M Tb(III) chelate, 103 M K2S2O8.
times. The treatment with the former seemed to have only a meager effect on HECL-emission intensity. When electrodes were pretreated with concentrated sulfuric acid the change in emission intensity was more drastic, more than a hundredfold decrease in emission intensity was observed when the electrodes were soaked in sulfuric acid for ten minutes before the HECL measurement. This can be explained by the partial removal of the polystyrene layer, which is supported by the fact that increase in acid treatment time seemed to increase the number of pulses it took to obtain highest emission intensity due to more unexposed graphite particles present on the electrode surface. The observed decrease in the emission intensity with both sodium hydroxide and sulfuric acid pretreatments gives further validation for the importance of the insulating layer with the present composite electrodes. 3.6. M/PG electrodes under DC polarization When direct current (DC) is used to drive the electron tunneling process with aluminum electrodes the insulating layer is disintegrated within milliseconds when voltage is applied to the electric circuit [29]. Quite similarly, when DC excitation was used with the M/PG electrodes the light emission was observed only for a couple of seconds before a sudden crash in the emission intensity (Fig. 6, curve b), after which the emission intensity abruptly increased and stayed at the same level for rest of the measurement. The steep decrease in emission intensity is caused by a mechanical failure of the PG composite that was observed to be detached from the brass substrate. However, a thin layer of PG composite still has to exist on the brass surface that makes the hot electron emission possible. The violent detachment of the composite is likely caused by the generation of hydrogen gas/bubbles in the vicinity of the composite/brass interface, which was not observed with pulse polarization due to less charge being transported and the periodical nature of charge injection. Thicker PG electrodes were spin coated which helped to alleviate this problem completely (Fig. 6 curves a and c). The steady decrease in emission intensity during measurement is observed both for Tb(III) chelate/S2O2 8 -system and S2O28 -blank and therefore likely related to consumption of peroxide which is known to increase the blank emission in HECL [30]. Another ill effect caused by the vigorous gas formation during the DC excitation is the noisier nature of the emission intensity signal, which coupled with the inability to perform time resolved
5
Fig. 6. DC excitation, electrodes (wg ¼ 30%) made from inks containing 50 (b) or 200 (a,c) g L1 polystyrene. 104 M Tb(III) chelate, 103 M K2S2O8 (curves a,b) and blank (c).
measurements makes the detection limits considerably worse. On the other hand, the use of DC excitation simplifies the instrumentation even further. 3.7. M/PG electrodes as studied by cyclic voltammetry Cyclic voltammetry (CV) was used to analyze the surface of the glassy carbon/PG electrodes (Fig. 7), i.e. to determine if the electrodes with varied composition would show active or passive behavior. If one electron reduction of ferricyanide to ferrocyanide takes place on the graphite surface the measured peak current is governed by Randles-Sevcik relationship which states that peak current is linearly dependent on electroactive surface-area. In order for electron transfer to occur, the energy levels of the donor states within the carbon and the acceptor levels within the solution must be in resonance. The voltammogram for wg ¼ 30% electrode shows that the surface of the M/PG composite is passivated, i.e. graphite particles are not in direct contact with the electrolyte solution, which makes the recorded faradaic current non-existent. As shown
Fig. 7. CV for various electrodes. Uncoated glassy carbon (solid line), glassy carbon electrode coated with graphite/polystyrene mixture containing 30% (dashed line), 50% (dotted line) and 55% (dot dashed line) of graphite. 100 mV s1 scan rate in 1 mM [Fe(CN)6]4 solution with 1 M KCl as the supporting electrolyte.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
6
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
previously in Fig. 1B the light forming chemical reactions are taking place practically near the whole cathode surface. Area this large should have been effortlessly detectable by CV if it was not covered by polystyrene layer, as the average distance it takes electron to localize as well as the average distance they drift-diffuse before reacting with water is estimated to be 2.5 ± 1.0 nm [31]. At 55% graphite concentration a clear oxidation peak was observed at 0.3 V (vs. SCE), but the charge transfer kinetics are very slow as seen from the high DEp-value, suggesting that a degree of electrical insulation around the graphite flakes exists even at higher graphite flake values. The different electrochemical response of electrodes shown in Fig. 7 complements results shown in Figs. 1 and 3. Bare graphite flakes are undoubtedly present on the electrolyte interface after M/PG electrodes contain more than 53% of graphite and the initial mechanism of charge transport during cathodic pulse polarization is normal electrochemical reduction taking place directly on the cathode surface. It is likely that bare graphite flakes are present at the electrode/electrolyte interface even at lower graphite concentrations as the observed emission intensity started to decline after Wg reached values beyond 30%. However, if this is the case, the amount of bare graphite has to be small as the CV measurements lacked the oxidation peak for electrodes containing 50% of graphite. In the light all these measurements it seems that the electron injection from M/PG electrodes is based on electron tunnel emission trough insulating layer rather than some kind of field emission from bare graphite flakes. 3.8. Analytical applicability of M/PG electrodes Log-log calibration curve for the blank-corrected Tb(III) chelate/ S2O2 8 -system is shown in Fig. 8 for both DC (squares) and pulse polarization excitation (circles). Good correlation between Tb(III) chelate concentration and measured TR-HECL signal was obtained in both cases, with pulsed excitation the error bars at higher chelate concentrations are smaller than the data points themselves. The detection limit (s/n ¼ 3, rounded up to the nearest power of ten) was 109 M for pulse polarization and 106 M with DC excitation. Presented in the inset of Fig. 8 are the decay profiles for blank and nanomolar Tb(III) chelate emissions. The amount of excitation
Fig. 8. Blank corrected calibration curve of Tb(III) chelate/103 M S2O2 8 -system with pulsed- (filled circles) or DC- excitation (empty squares), wg ¼ 30 %for both cases, n ¼ 7 for each data point. Inset: decay of emission intensity, blank (b) and 109 M Tb(III) chelate (a).
pulses was triple the amount used in the standard curve measurements to make the difference between the blank and sample emissions easier to observe visually. The difference in the luminescence lifetimes allows for effortless distinction between these two signals when HECL is measured by time-resolved technique. In HECL the following factors are affected by the working electrode: the amount of hydrated electrons generated (i.e. intensity of TR-HECL signal), intrinsic electroluminescence from the electrode material (noise that affects the s/n-value) and the reproducibility of TR-HECL signal between electrodes. Repeatability was examined by measuring seven disposable electrodes. The standard deviation for the measured Tb(III) chelate emission at 105 M concentration was 4.3% when the excitation was done with pulse polarization and roughly 23% when DC excitation was used. This shows that the performance of M/PG electrodes is sufficiently good to warrant further research. 3.9. Blank emission under cathodic polarization Electroluminescence (EL) is the main contributor to blank signal in HECL. With thin insulating film-coated electrodes the EL is mainly based on impact excitation of luminescent impurities and on electron center luminescence (F-center, E-center, etc.) at the insulator/electrolyte interface [32]. The lowest background emission is obtained when the oxide film does not contain any luminescent impurities and encloses as low number of defect states as possible. Because the polystyrene can be mixed with variety of different conducting particles, each having a unique EL fingerprint, the EL was measured for pure polystyrene thin-films and for M/PG composite electrodes (Fig. 9). The pure polystyrene films were spin coated from solution containing only 5 g L1 polystyrene. With these metal/polystyrene-electrodes the short-lived electroluminescence appears only during the cathodic potential pulse and fades away instantly when the excitation pulse ends (Fig. 9 curves c and d). For M/PG electrodes (wg ¼ 30%) there is clearly long-lived luminescence of some origin (Fig. 9 curves a and b) that lowers the s/n-ratio considerably, even when optical filtration is used and when the luminophore has a long luminescence lifetime. The purity of graphite flake powder (99.53%) was measured with X-ray fluorescence analysis, impurities included silicon, phosphorous, aluminum, sulfur and less than 0.05% of Ca, Mg, Cu and Pb.
Fig. 9. Decay of electroluminescence in borate buffer solution. Plotted with respect to the end of the excitation pulse. M/PG electrode (wg ¼ 30%) without (a) and with optical filter (b). Pure polystyrene thin film without (c) and with a filter (d). 4000 excitation pulses.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035
K. Salminen et al. / Analytica Chimica Acta xxx (2017) 1e7
4. Conclusions Hot electron-induced electrochemiluminescence of Tb(III) chelate/S2O28 -system in aqueous solutions at metal/polystyrenegraphite composite electrodes was investigated for the first time. The excitation of the aromatic Tb(III) chelate was mainly based on the injection of hot electrons into electrolyte solution from the graphite particles embedded in the polystyrene matrix. Due to the generation of solvated electrons the metal/polystyrene-graphite composite electrodes are capable of exciting other luminophores such as Ru(bpy)2þ 3 as well [33]. The observed emission intensity was profoundly dependent on the amount of graphite particles present in/on the composite material. The graphite weight fraction range at which the electrodes were capable of exciting the Tb(III) chelate was very wide: 1e65%. The insulating polystyrene layer at the electrolyte interface seemed to be necessary for the efficient generation of hydrated electrons and subsequent electrochemiluminescence. Detection limit for Tb(III) chelate was ca. 109 M with pulse- and 106 M with DC-excitation. When more suitable conducting particles than presently used are applied, polystyrene based composite electrodes can be suggested to fully replace silicon disc electrodes in low-cost disposable plastic cartridges, making it reasonable to use silicon electrodes only in those cases when sophisticated electrode chips are used and fabricated in sufficiently large batches [15,34]. Funding Preliminary experiments of these studies were supported by SalWe Oy/Technology development center of Finland (534/14). References [1] M.J. Bronskill, Picosecond pulse radiolysis studies. I. The solvated electron in aqueous and alcohol solutions, J. Chem. Phys. 53 (1970) 4201e4210, http:// dx.doi.org/10.1063/1.1673922. [2] P. Cintas, J.-L. Luche, Green chemistry. A sonochemical approach, Green Chem. 1 (1999) 115e125, http://dx.doi.org/10.1039/a900593e. [3] J. Goodman, A. Hickling, B. Schofield, The yield of hydrated electrons in glowdischarge electrolysis, J. Electroanal. Chem. Interfacial Electrochem 48 (1973) 319e322, http://dx.doi.org/10.1016/S0022-0728(73)80272-4. [4] F. Tochikubo, Y. Shimokawa, N. Shirai, S. Uchida, Chemical reactions in liquid induced by atmospheric-pressure dc glow discharge in contact with liquid, Jpn. J. Appl. Phys. 53 (2014) 126201, http://dx.doi.org/10.7567/JJAP.53.126201. [5] M.S. Matheson, W.A. Mulac, J. Rabani, formation of the hydrated electron in the flash photolysis of aqueous solutions 1, J. Phys. Chem. 67 (1963) 2613e2617, http://dx.doi.org/10.1021/j100806a027. k, T. Buttersack, S. Bauerecker, P. Jungwirth, [6] P.E. Mason, F. Uhlig, V. Vane Coulomb explosion during the early stages of the reaction of alkali metals with water, Nat. Chem. 7 (2015) 250e254, http://dx.doi.org/10.1038/ nchem.2161. [7] S. Kulmala, J. Suomi, Current status of modern analytical luminescence methods, Anal. Chim. Acta 500 (2003) 21e69, http://dx.doi.org/10.1016/ j.aca.2003.09.004. [8] S. Kulmala, A. Kulmala, T. Ala-Kleme, J. Pihlaja, Primary cathodic steps of electrogenerated chemiluminescence of lanthanide(III) chelates at oxidecovered aluminum electrodes in aqueous solution, Anal. Chim. Acta 367 (1998) 17e31, http://dx.doi.org/10.1016/S0003-2670(98)00154-8. [9] S. Kulmala, T. Ala-Kleme, L. Heikkil€ a, L. V€ are, Energetic electrochemiluminescence of (9-fluorenyl)methanol induced by injection of hot electrons into aqueous electrolyte solution, J. Chem. Soc. Faraday Trans. 93 (1997) 3107e3113, http://dx.doi.org/10.1039/a702135f. [10] M. Håkansson, Q. Jiang, M. Helin, M. Putkonen, A.J. Niskanen, S. Pahlberg, T. Ala-Kleme, L. Heikkil€ a, J. Suomi, S. Kulmala, Cathodic Tb(III) chelate electrochemiluminescence at oxide-covered magnesium and n-ZnO: Al/MgO composite electrodes, Electrochim. Acta 51 (2005) 289e296, http://dx.doi.org/ 10.1016/j.electacta.2005.04.033. [11] T. Ala-Kleme, S. Kulmala, Q. Jiang, Generation of free radicals and electrochemiluminescence from simple aromatic molecules in aqueous solutions, Luminescence 21 (2006) 118e125, http://dx.doi.org/10.1002/bio.895. [12] V.V. Shmanai, T.A. Nikolayeva, L.G. Vinokurova, A.A. Litoshka, Oriented antibody immobilization to polystyrene macrocarriers for immunoassay modified with hydrazide derivatives of poly(meth)acrylic acid, BMC Biotechnol. 1 (2001) 4, http://dx.doi.org/10.1186/1472-6750-1-4.
7
[13] E. Ishikawa, Y. Hamaguchi, M. Imagawa, M. Inada, H. Imura, N. Nakazawa, H.O. Daiichi, An improved preparation of antibody-coated polystyrene beads for sandwich enzyme immunoassay, J. Immunoass. 1 (1980) 385e398, http:// dx.doi.org/10.1080/01971528008058479. [14] S. Kulmala, M. Håkansson, A.M. Spehar, A. Nyman, J. Kankare, K. Loikas, T. AlaKleme, J. Eskola, Heterogeneous and homogeneous electrochemiluminoimmunoassays of hTSH at disposable oxide-covered aluminum electrodes, Anal. Chim. Acta 458 (2002) 271e280, http://dx.doi.org/10.1016/ S0003-2670(02)00065-X. [15] T. Ylinen-Hinkka, A.J. Niskanen, S. Franssila, S. Kulmala, Immunoassay of Creactive protein by hot electron induced electrochemiluminescence using integrated electrodes with hydrophobic sample confinement, Anal. Chim. Acta 702 (2011) 45e49, http://dx.doi.org/10.1016/j.aca.2011.06.038. [16] J.O. Koskinen, J. Vaarno, N.J. Meltola, J.T. Soini, P.E. H€ anninen, J. Luotola, M.E. Waris, A.E. Soini, Fluorescent nanoparticles as labels for immunometric assay of C-reactive protein using two-photon excitation assay technology, Anal. Biochem. 328 (2004) 210e218, http://dx.doi.org/10.1016/ j.ab.2004.02.029. [17] X.Y. Chen, R.J. Zheng, S.F. Qin, J.J. Sun, Hot electron-induced cathodic electrochemiluminescence at oil film-covered carbon paste electrode and application to nano-molar determination of catechol, Talanta 101 (2012) 362e367, http://dx.doi.org/10.1016/j.talanta.2012.09.042. [18] G.V. Buxton, C.L. Greenstock, W.P. Helman, A.B. Ross, Critical Review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals (,OH/,O) in Aqueous Solution, J. Phys. Chem. Ref. Data 17 (1988) 513e886, http://dx.doi.org/10.1063/1.555805. [19] S. Suomi, J. Kulmala, Reviews in Fluorescence 2009, Springer-Verlag, New York, 2009, http://dx.doi.org/10.1007/s10895-005-2528-0. [20] A.G. Krivenko, N.S. Komarova, E.V. Stenina, L.N. Sviridova, V.A. Kurmaz, A.S. Kotkin, V.E. Muradyan, Electrochemical behavior of electrodes containing nanostructured carbon of various morphology in the cathodic region of potentials, Russ. J. Electrochem 42 (2006) 1047e1054, http://dx.doi.org/10.1134/ S1023193506100090. [21] A.G. Krivenko, N.S. Komarova, Electron injection from nanostructured carbon electrodes at moderate cathodic potentials, Russ. J. Electrochem 43 (2007) 1123e1126, http://dx.doi.org/10.1134/S1023193507100035. [22] A.G. Krivenko, N.S. Komarova, P.N.P, Electrochemical generation of solvated electrons from nanostructured carbon, Electrochem. Commun. 9 (2007) 2364e2369, http://dx.doi.org/10.1016/j.elecom.2007.07.004. [23] P. Kuosmanen, K. Salminen, M. Pusa, T. Ala-Kleme, S. Kulmala, Hot electroninduced electrogenerated chemiluminescence of Ru(bpy)2þ chelate at a 3 pointed active metal cathode in fully aqueous solutions, J. Electroanal. Chem. 738 (2016) 63e67, http://dx.doi.org/10.1016/j.jelechem.2016.10.057. [24] T. Ylinen, J. Suomi, M. Helin, T. Ala-Kleme, S. Kulmala, Time-resolved detection of hot electron-induced electrochemiluminescence of fluorescein in aqueous solution, J. Fluoresc. 16 (2006) 27e33, http://dx.doi.org/10.1007/s10895-0050023-2. [25] S. Kulmala, K. Haapakka, Mechanism of electrogenerated luminescence of terbium(III)-“2,6-bis[N,N-bis(carboxymethyl)aminomethyl]-4benzoylphenol” chelate at an oxide-covered aluminium electrode, J. Alloy. Compd. 225 (1995) 502e506, http://dx.doi.org/10.1016/0925-8388(94) 07058-X. [26] S. Nakamura, K. Saito, G. Sawa, K. Kitagawa, Percolation threshold of carbon black-polyethylene composites, Jpn. J. Appl. Phys. Part 1 Regul. Pap. Short. Notes Rev. Pap. 36 (1997) 5163e5168, http://dx.doi.org/10.1143/JJAP.36.5163. [27] J. Li, J.K. Kim, Percolation threshold of conducting polymer composites containing 3D randomly distributed graphite nanoplatelets, Compos. Sci. Technol. 67 (2007) 2114e2120, http://dx.doi.org/10.1016/j.compscitech.2006.11.010. [28] M. Salovey, R. Ghofraniha, Electrical conductivity of polymers containing carbon black, Polym. Eng. Sci. 28 (1988) 58e63, http://dx.doi.org/10.1002/ pen.760280110. [29] M. Håkansson, Q. Jiang, A.M. Spehar, J. Suomi, M. Kotiranta, S. Kulmala, Direct current-induced electrogenerated chemiluminescence of hydrated and chelated Tb(III) at aluminum cathodes, Anal. Chim. Acta 541 (2005) 171e177, http://dx.doi.org/10.1016/j.aca.2004.12.072. [30] S. Kulmala, T. Ala-Kleme, A. Hakanen, K. Haapakka, F-Centre luminescence from oxide-covered aluminium cathode induced by two-step reduction of peroxydisulfate anions, J. Chem. Soc. Faraday Trans. 93 (1997) 165e168, http://dx.doi.org/10.1039/a604025j. [31] P. Rumbach, D.M. Bartels, R.M. Sankaran, D.B. Go, The solvation of electrons by an atmospheric-pressure plasma, Nat. Commun. 6 (2015) 7248, http:// dx.doi.org/10.1038/ncomms8248. [32] M. Håkansson, Q. Jiang, J. Suomi, K. Loikas, M. Nauma, T. Ala-Kleme, J. Kankare, P. Juhala, J.U. Eskola, S. Kulmala, Cathodic electrochemiluminescence at double barrier Al/Al2O3/Al/Al2O3 tunnel emission electrodes, Anal. Chim. Acta 556 (2006) 450e454, http://dx.doi.org/10.1016/j.aca.2005.09.064. [33] T. Ala-Kleme, S. Kulmala, L. Vare, P. Juhala, M. Helin, Hot electron-induced electrogenerated chemiluminescence of Ru(bpy)2þ 3 chelate at oxide covered aluminum electrodes, Anal. Chem. 71 (1999) 5538e5543, http://dx.doi.org/ 10.1021/ac981336i. [34] M. Håkansson, K. Salminen, P. Kuosmanen, J. Eskola, H. Peuravuori, S. Kulmala, Immunoassay of b2-microglobulin at oxide-coated heavily doped p-silicon electrodes, J. Electroanal. Chem. 769 (2016) 11e15, http://dx.doi.org/10.1016/ j.jelechem.2016.02.040.
Please cite this article in press as: K. Salminen, et al., Cathodic electrogenerated chemiluminescence of aromatic Tb(III) chelates at polystyrenegraphite composite electrodes, Analytica Chimica Acta (2017), http://dx.doi.org/10.1016/j.aca.2017.07.035