Electro-Photodynamic Visualization of Singlet Oxygen Induced by Zinc

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Electro-Photodynamic Visualization of Singlet Oxygen Induced by Zinc Porphyrin Modified Microchip in Aqueous Media Chuanguang Yao, Hongxin Song, Ying Wan, Kefeng Ma, Chenyu Zheng, Hongda Cui, Peng Xin, Xubo Ji, and Sheng-Yuan Deng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 20 Oct 2016 Downloaded from http://pubs.acs.org on November 11, 2016

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ACS Applied Materials & Interfaces

Electro-Photodynamic Visualization of Singlet Oxygen Induced by Zinc Porphyrin Modified Microchip in Aqueous Media Chuanguang Yao,1,† Hongxin Song,1,† Ying Wan,2,* Kefeng Ma,1 Chenyu Zheng,1 Hongda Cui,1 Peng Xin,1 Xubo Ji,1 Shengyuan Deng1,3,* 1

2

School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing 210094, P.R. China Intelligent Microsystem Technology and Engineering Center, School of Mechanical Engineering, Nanjing University of Science and Technology, Nanjing 210094, P.R. China

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Abstract A porphyrin-based electro-photodynamic imaging system was fabricated for monitoring the concentration of oxygen. Distinct from the electrochemiluminescent (ECL) inability of numerous organic species in aqueous solutions, a strong and stable red irradiation at 634 nm could be stimulated electrochemically on zinc(II) meso-tetra(4-carboxyphenyl) porphine (ZnTCPP)/tetraoctylammonium bromide (TOAB) in the physiological condition. In terms of in situ electron paramagnetic resonance and ECL spectroscopies, the nature of ECL was thoroughly investigated, being exactly the chemiluminescence from singlet oxygen (1O2) produced during the successive electro-reduction of ZnTCPP. Meanwhile, the excellent film-making capacity of amphiphilic TOAB as a potent ion barrier granted the luminophores a micro-order and patternable electrode modification. Such platform was exceptionally tolerant of pH variation, facilitating a durable solid-state ECL visualization under potentiostatic electrolysis and time exposure in the charge-coupled device (CCD) camera. For flow-injection and real-time detection, a chip-mounted microfluidic cell was customized and manufactured. A sensitive and simple vision-sensing of O2 was further achieved with a real determination limit as low as a few micromolar level. The developed ECL imaging system is the first and an eco-friendly case in the cathodic range, thus would supplement the primary anodic imaging library, showing great promise in multiplexed and colorimetric assays as well as oxygen-involved activity studies in future. Keywords: Electrochemiluminescent imaging; Zinc porphyrin; Tetraoctylammonium bromide; Singlet oxygen; Microchip ______________________________ * Corresponding author. E-mail: [email protected], [email protected]. † These authors contributed equally to this work.


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1. INTRODUCTION Owing to the electroexcitation nature, visualized electrochemiluminescence (ECL) techniques have predominance to the analytical interest in both ultrahigh detectability out of a pitch-black background and spatio-temporal modulability in terms of electron-transfer reactions.1 To date, resolutions at single cell, nanoparticle and picoliter droplet levels have all been accomplished by ECL microscopic imaging;2,3 while scanning probe-hypenated fingerprint identification,4 prototypes of flexible electronic devices,5,6 as well as intelligent integrated liquid circuits and lab-on-chips,7,8 enormously expanded the related realistic practices. In the very essence, these remarkable progresses benefitted from the exploration and exploitation of highly efficient ECL luminophores, mainly the ruthenium tris(bipyridyl)-derived complexes and composites.9 Picking some biosensing examples, arrays for screening metabolite-generated toxicity utilizing spots containing a fully representative set of metabolic enzymes from microsome and cytosol in both human and rat livers and ECL polymer ([Ru(bpy)2PVP10]2+) were eyeballed with a CCD camera;10 what is more, traditional ECL intensity information of tripropylamine, proline, and dopamine have all been transformed sucessfully into a color variation by fabricating a dual-color system: a red source Ru(bpy)32+ and a green light emitting diode as the reference.11 On the other hand, although numerous polycyclic and heterocyclic aromatic hydrocarbons, along with the boron dipyrromethene (BODIPY) dye series, have also highlighted their ECL potencies,12,13 few of them could be reproduced in the aqueous solvents due to insolubility and radical instability plus the narrow potential window of water,14 which is on the contrary uniquely favored by bioimaging. Thus the development of versatile strategies based on the ECL imaging and other proof-of-concept implementations are severely restricted, especially



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for the arrayed, multiplexed, colorimetric, and ratiometric assays. To overcome such issues, several trials have been purposed, for example, condensed nanocrystals kept molecular or polymeric emitters inside from protons,15 and surfactants incorporated diphenylanthracene into nanomicelle that was mass-transferred hydrodynamically towards the electrode.14 Recently, Bard et al. maneuvered the collision event of tiny emulsion reactors,16 and observed the ECL emission from rubrene within in an aqueous continuum.3 However, the above scenarios focused on the elaborate fabrication of liquid models, short in the intensity and inadaptable to interfacial immobilization and probe tagging. Needless to say, despite the polychroism of quantum dots (QDs) by the intrinsic size effect,17 nobody could deny the fact that their generally low ECL yield and unsuppressed blinking behavior can hardly qualify for powerful and persistent electrochemical light sources.18 In view of these, it is a universal knowledge that, prior to the imaging analysis, a facile and flexible solid-state ECL mode of organic species should be realized in the physiological condition. Here, inspired by those published works, one simple rule for the organic ECL appearance in the aqueous regime was assumed as evading the electrophilic attacks from protons in water, and was utilized by employing the amphiphilic tetraoctylammonium bromide (TOAB) as a waterproof fabric.19 A strong and stable ECL irradiation could then be achieved in pH 7.0 buffered solutions from zinc(II) meso-tetra(4-carboxyphenyl)porphine (ZnTCPP) embedded in TOAB modified electrode (Scheme 1), which was significantly different from the sparsely reported ECL ″rarity″ of divalent metalloporphyrins (i.e., copper, palladium, ruthenium) in degassed and/or aprotic phases.20 Our previous discovery on the ECL machinery of zinc(II) proto-porphyrin IX also pushed us forward to surmount such aquatic difficult of ECL incapacitation.20 More than that, the dissolved oxygen (DO) was verified ready to fuel sufficient excitons in the process, emerging as perceptible and permanent spots in a digit 4 ACS Paragon Plus Environment

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camera, which turned out to be the CL of singlet oxygen (1O2), the lowest energy excited electronic state of molecular oxygen, during the electro-reduction of ZnTCPP. A novel ″electro-photodynamic″ mechanism was hence established via in situ electron paramagnetic resonance (EPR), theoretically analogous to that of photodynamic therapy.21 Besides, the excellent film-making capacity of TOAB could facilitate a homogeneous and patternable layer on the tailored screen-printed electrode with a broadened and sensitized electrochemical response,22 regardless of the variation in pH. As O2 a crucial participant in a wide range systems from oxygenation reactions of polymers to cell necrosis and cancer diseases,23,24 a real-time and ultrasensitive monitoring of trace amount of O2 was visualized in a chip-mounted microfluidic vessel based on the principle of emitter production (Scheme 1). Taking the merit of high

1

O2 quantum yields without photobleaching, this compact

TOAB/ZnTCPP combination exemplifies the easy preservation and sustainability of ECL from organo-molecules in various media, stepping into the breach of cathodic imaging, which would exert a profound influence upon the conventional ECL applications.

2. EXPERIMENTAL SECTION 2.1. Chemical Reagents. Tetraoctylammonium bromide (TOAB), 5,5-dimethyl-1pyrroline N-oxide (DMPO, sealed in dry ice), N-(2-Hydroxyethyl)piperazine-N′-(2ethanesulfonic acid) (HEPES) sodium salt, 2,2,6,6-tetramethylpiperidine (TMP), anhydrous dichloromethane (CH2Cl2 or DCM, contains 50~150 ppm amylene as stabilizer), sodium azide (NaN3), Rose Bengal (dye content 95%), disodium 9,10-anthracendipropionic acid (ADPA), superoxide dismutase (SOD, E.C. 1.15.1.1, from bovine erythrocytes, 4200 units·mg−1 solid), and tetrabutylammonium percholate ((n-C4H9)4N+∙ClO4− or TBAP) were purchased from 5 ACS Paragon Plus Environment


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Sigma-Aldrich Chemical Co., Ltd. (Shanghai, China). Zinc(II) meso-tetra(4-carboxyphenyl) porphine (ZnTCPP), meso-tetra (4-carboxyphenyl)porphyrin (TCPP), L-cysteine hydrochloride, and toluene were procured from J&K Chemical Ltd. (Shanghai, Chia). All other reagents were of analytical grade and used as received. Ultrapure water from a Millipore purification system (≥18 MΩ, Milli-Q) went throughout all assays. The detection solution was 10 mM pH 7.4 HEPES buffer saline prepared by spiking the stock solutions of 10 mM HEPES acid and 10 mM HEPES in NaOH, both containing 0.3 M KCl as the supporting electrolyte. 0.1 M TBAP as the supporting electrolyte in DCM was specialized in spectroscopic experimentation. The O2 or N2-saturated solution was premade by constantly bubbling pure O2/N2 to preserve the atmospheric pressure. 2.2. Apparatus. The morphology was probed with an FEI Quanta 250F field emission scanning electron microscope (FESEM) (Houston, USA) operated at an accelerating voltage up to 15 kV. Thin-film X-ray diffractograms of both small-angle scattering and wide-angle diffraction were finished on a D8 ADVANCE low-temperature X-ray diffractometer (Bruker, Germany) with Kα X-rays of Cu (λ = 1.54 Å) from a Bruker generator. UV-Vis absorption spectra were recorded on a UV−3600 UV-Vis-NIR spectrophotometer (Shimadzu Co., Japan). Steady-state photoluminescence (PL) spectra along with the ECL spectrum were collected by an Edinburgh FLS920 fluorescence spectrometer (Livingston, UK). Cyclic voltammetry (CV) and square wave voltammetry (SWV) measurements were performed using a CHI 660D electrochemical workstation (Chenghua Instruments Inc., China). The ECL imaging was photographed after programmed continuous exposure in a Tanon−5200 Multi automatic chemiluminescence/fluorescence transilluminator (Tanon Co., Ltd., China) with a cooled low-light high-resolution charge-coupled-device (CCD) camera. Both ECL spectroscopy and imaging were synchronized with a CHI 660D electrochemical workstation by galvanizing at 6 ACS Paragon Plus Environment

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−1.7 V via hydrodynamic potentiostatic electrolysis, e.g. amperometric i~t curve. A Windows 7 script was compiled for timing startups in succession of dual programs. In situ electron paramagnetic resonance (EPR) spectra were instantaneously hyphenated on an EMX−10/12 EPR spectrometer (Bruker Co.) with electrochemical instrumentation and 430 nm incident laser. The ECL kinetic measurements were carried out on a MPI−EII multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remex Analytical Instrument Co., Ltd., China) in which the emission window (detection range: 280~850 nm) was right above the photomultiplier tube (PMT) biased at −800 V with two-fold magnification. Standard three-electrode protocol was abided by all equipment, including the screen-printed electrode (SPE). For preliminary tests, glassy carbon electrode (GCE, 5 mm in diameter) was polished to a mirror finish mechanically by 0.3 and 0.05 μm alumina powders. The well-polished GCE was sequentially cleaned in absolute ethanol and deionized water by sonication for 1 min, separately. The reference (a Ag/AgCl with saturated KCl as filling liquid in a Luggin capillary) and counter (a platinum wire) electrodes were plugged in each side of a home-made cell, while the modified GCE as working electrode was placed in the middle nozzle with its surface downward abut upon the optical window to enlarge the signal collection. A branch slot was left over for ventilation and injection. All potentials were quoted against this reference. Unless specifically mentioned, the scan rate was 50 mV s−1. 2.3. Preparation of TOAB/ZnTCPP Modified SPE. As the protic alcohols can provide the protons needed to ligate with the imidazolyl nitrogen of porphine ring, while toluene does not,25 which was utilized as nonsolvent instead of short-chain hydroxyls (e.g. methanol, ethanol and iso-propyl alcohol) during the cosolubilization of ZnTCPP and TOAB. Typically, ZnTCPP (2.0 mg, 2.4 mM) and TOAB (10.9 mg, 20 mM) were sonicated in 1.0 mL toluene for 30 min and repeated for 4 times. After that, 20.0 μL of the mixture was directly spread on the 7 ACS Paragon Plus Environment


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surface of cleaned GCE (10.0 μL for SPE, 5.0 μL of one pipetting) and dried at ambient temperature. For comparison, TOAB/GCE and TCPP/TOAB/GCE were fabricated under identical conditions. TOAB/GCE in 0.1 M pH 4.5 sodium acetate/acetic acid buffer solution containing 10 mM zinc acetate (Zn(Ac)2) was used to electrodeposit zinc (TOAB/Zn/GCE) at −1.6 V for 15 s. 2.4. 1O2 Quantum Yield Measurements. (1) Chemical Method: 300 μL of 2.4 mM Rose Bengal was employed as the standard photosensitizer and incubated with 1.2 mL of 1 mg·mL−1 ADPA in O2-saturated HEPES buffer under an incandescent lamp (400~800 nm, 6.5 mW·cm−2). In the ECL experiment, 500 μL of 1 mg·mL−1 ADPA as 1O2 trapper was added to 15 mL HEPES and poised to be consumed. In two cases, the UV absorptions of ADPA at 378 nm were itemized intermittently. To eliminate the inner-filter effect, the absorption maxima of Rose Bengal and ZnTCPP were adjusted to ~0.2 O.D. (optical density). The 1O2 quantum yield at TOAB/ZnTCPP/GCE (ΦZnTCPP) was deduced by:26 ΦZnTCPP = ΦRose Bengal × [κ / Λ]ZnTCPP / [κ / Λ]Rose Bengal

(1)

where κ and Λ are deemed as the decomposition rate of ADPA and the absorptive light (integration of optical absorption bands, 400~700 nm) by ZnTCPP and Rose Bengal, and ΦRose Bengal

= 0.75 as default.

(2) Emissive Method: The 1O2 emission of Rose Bengal was detected in an Edinburgh FLS920 fluorescence spectrophotometer with a 430-nm excitation laser and a near-infrared detector, while the ECL emission of 1O2 at TOAB/ZnTCPP/GCE was executed in the PL dark chamber with input wires connecting CHI 660D. Considering the short PL lifetime of 1O2 in water, the solid power of 2.0 mg ZnTCPP was imported in O2-saturated CH2Cl2/TBAP solution. The absorptions of Rose Bengal and ZnTCPP at 430 nm were also depressed to ~0.2


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O.D, and the ECL spectrum was demarcated by subtracting the self-fluorescent intensity of ZnTCPP. In the same way, ΦZnTCPP could be reckoned using:26 ΦZnTCPP = ΦRose Bengal × ΛZnTCPP / ΛRose Bengal

(2)

where Λ conveys the integral peak area of 1O2 in emission spectra, and ΦRose Bengal is known to be 0.76 in DCM. 2.5. Miniaturization of ECL Device. In detail, the sensing archetype was divided into two modules (Both were kindly gifted by Professor Ying Wan at Intelligent Microsystem Technology and Engineering Center): (1) A homemade rectangular cassette composed of polydimethylsiloxane (Mass ratio of monomer vs. elastomer: 10:1, Vacuum degassed and Aged at 60 °C overnight in an induction cooker) with a built-in flat cylindrical cavity of 100 μL, a inlaid watertight O-ring and one pair of inlet/outlet conduits (Width: 180 μm, Thickness: 100 μm) for programmed channeling from the analyte reservoir and gas pressurization (Scheme 1). A side slit on the dented wall of micro-well was preserved for the insertion of SPE, which could also be customized for a paralleled electrode array, depending on the demand.27 (2) An SPE modified with TOAB/ZnTCPP as the microchip, whose three-electrode mode was patterned with Adobe Illustrator CS6 of a carbon master as working electrode and a silver semicircle as the reference before committed to chrome-masked photoresist and lithography. This microfluidic cell could be switched on/off by an airproof cover. Ahead of field practices, the entire chip was mounted up into fitted accommodation with its luminous surface against the transparent quartz for a clear snapshot. 2.6. ECL Visualization of Dissolved Oxygen. Oxygen-carrying solutions with different concentrations were premixed in a thermal couple by syringing various volumes (max. 1.0 mL) of O2- saturated 10 mM pH 7.4 HEPES (Lane A) through tubing into deaerated 10 mM pH 7.4 HEPES (Lane B) following a cryo-thaw gradient cycle, whose flow rate was 9 ACS Paragon Plus Environment


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stabilized at 200 μL·hr−1 by a valve-gated digital barometer and a 4-channel syringe pump. The hybrid was automatically delivered to immerse the sensing site and paused for 2 min as preset as the space filled with 100 μL of shallow liquids. The ECL signal on the SPE could be simultaneously captured by hypothermal cooled CCD with a dynamic integration of 60 s, an interval of 30 s plus a full-length acquisition of 30 min (Scheme 1) and subsequently identified by Bioanalysis software (Tanon) and ImageJ picture acquisition. The ECL intensity was computed as the mean pixel (resolution of the acquired window: 1024×768, 16 bit, and the binning value: 4) intensity over each circular bright spot (1 pixel ≈ 1.3 μm). To ensure the homogeneity of batch processing, the reactor should be rinsed with highly pure N2-purged detection solution as fast as possible.

3. RESULTS AND DISCUSSION 3.1. Characterization of TOAB/ZnTCPP/SPE. The morphologies of ZnTCPP and TOAB/ZnTCPP modified SPE were characterized by FESEM. Individual ZnTCPP conglomerated and stacked into vesicle-like bodies of varying sizes from 0.1 to 0.5 μm on the surface of bare SPE (Figure 1A). After being enveloped in TOAB, which was fairly miscible in toluene, aggregation declined and ZnTCPP molecules were well-dispersed as a uniform layer (Figure 1B). The high viscosity of this blend made it easy to shape edge contours during the evaporation of toluene at ambient temperature. As a matter of fact, TOAB/ZnTCPP possessed an incredible subsurface crystallinity as patterned in Figure 1B inset, which is speculated to be a packing arrangement of discrete domains in the solid phase.28 The strong, low-angle summit in degree (2θ = 4.77) of the diffractogram was converted to the distance (d = 18.49 Å) using Bragg’s equation (λ = 2d·sinθ).29 The ionic radius of Br− (1.82 Å) and its electrostatic


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attraction with N+ plus the splaying out of three octyl chains, translated to such capacious lamellar spacing allocated to every TOAB that would accommodate a prophyrin molecule (Lateral Length: 14.39 Å, from its Solvent Accessible Surface Area: ~207 Å2).30 This homogeneous interfacial structure were favorable for promoting the electron shuttling of ZnTCPP and facilitating the access of analytes. Prior to the manifestation of ECL features, its synchronous stimulation by electricity was investigated by cyclic voltammograms (CVs) of different modified electrodes in air-free 0.5 M KCl solution. On neither TOAB/GCE nor ZnTCPP/GCE could peaks be found except flat base currents in the potential window between −1.7 and 1.0 V (Figure 2A, curves a and b), indicating inactive electrochemistry of TOAB and ZnTCPP in water solution. In stark contrast, by combining the both, three obvious pairs of seemingly quasi-reversible redox peaks, designated as couples I, II, IV from negative to positive, appear clearly for TOAB/ZnTCPP/ GCE (Figure 2A, curve c), the formal potentials [(Epreduction+Epoxidation)/2] of which were estimated to be −1.57, −1.07 and 0.835 V, respectively. The shape of the plot tended to be stable, implying that no structural change has taken place in the TOAB/ZnTCPP membrane ever since the first start of electro-reduction.31 These reduction peak currents (ipc) were validated to be proportional to the square root of scan rates from 10 to 100 mV·s−1 (Figure 2B inset), suggesting that the heterogeneous electron transfer of solid-state ZnTCPP actually follows a diffusion-controlled process. This deduction fits the prediction about macroscopic self-assembly of TOAB (Figure 1B inset) and is in good agreement with the redox behavior of artificial p-methoxy porphyrin−C60 clusters inside amphiphiles.22 In order to chemically identify those peak belongings, the electrochemical processes of TOAB/ZnTCPP/GCE and TOAB/TCPP/GCE were studied together. By means of SWV (Figure 2B), the electro-activity related to zinc ligation in ZnTCPP was probed by stripping 11 ACS Paragon Plus Environment


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preconcentrated elemental zinc precipitation (deposited at −1.6 V for 15 s) on TOAB/Zn/GCE in anaerobic condition. Double baseline-resolved pairs (I and II) stuck out with formal potentials of −1.67 and −0.91 V, which were assigned to Zn0/•+ and Zn•+/2+ (Figure 2B, curve b),32 and corresponded to zincous valence-shell electronic contribution to the highest occupied molecular orbital of ZnTCPP, that is, Peak I (Zn0/•+TCPP, −1.57 V) and Peak II (Zn•+/2+TCPP, −1.06 V) at TOAB/ZnTCPP/GCE (Figure 2A, curve c and Figure 2B, curve a), respectively. The standard potentials of concerned half-reactions were listed below (e.g. Eq. (3) and (4)): Zn2+TCPP + e− → Zn•+TCPP

(Ep = −1.06 V)

(3)

Zn•+TCPP + e− → Zn0TCPP

(Ep = −1.57 V)

(4)

As for TOAB/TCPP/GCE, four peaks locate cathodically at −0.73, −0.93, −1.19 and −1.37 V (from I to IV in Figure 2A inset). By analogy with the direct electrochemistry of porphyrins, diprotonated TCPP underwent a progressive four-electron reduction: [H+]2TCPP2+ → [H+]TCPP•+ → TCPP0 (unsubstituted) → [H]TCPP•− → [H]2TCPP2−.33 Especially, its peak at −1.37 V drew close to the shoulder at −1.45 V of ZnTCPP, indicating the generation of transient meta-state Zn•+TCPP•− preceded the electron-injection of Zn0TCPP and the hydrogen evolution. Stereochemically comparable to methemoglobin and oxyhemoglobin,34 the wide Peak III in Figure 2B is attributed to the electro-dehydroxylation of central zinc (HO−Zn2+TCPP → O=Zn2+TCPP), from four-ligated sp2d hybridization (formation constant: logKf ~29.0) to distorted sp3d2-type. As can be seen, TCPP reveals only one anodic peak at ~0.8 V (Figure 2A, curve d), the oxidation peak potential (Epa) of which is a little negative than Peak IV of ZnTCPP and in accordance with the result of D'Souza et al.35 In this way, the distinguished Peak IV was ascribed to the overlap of redox of Zn2+TCPP0/∙+ and Zn2+TCPP∙+/2+.22 The symmetry of both Peak III and IV were contingent on pH and the solvation effect.36 Noteworthily, no more peaks 12 ACS Paragon Plus Environment

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could be recognized in comparison with the refined voltammetric characteristics of porphyrinogen homologues in o-dichlorobenzene,22 which may be interpreted as extra easy electron relay of ZnTCPP in high-conductance ionic liquid crystals of quaternary ammonium/phosphonium halides,37 leading to the superposition in Peak IV. Therefore, TOAB/ZnTCPP as a substrate is desirable to serve as an electron-mediating entity for the fabrication of relevant devices. 3.2. Aqueous ECL Capability of TOAB/ZnTCPP. As shown in Figure 3A, without being amalgamated with TOAB, ZnTCPP itself though very capable of ECL in several organic solvents, including the typical dipolar DCM (Figure 3A, curve a), failed to emanate any light in almost the same conditions except for the aqueous solution (Figure 3A, inset b). This drastic inconsistence could not be given rise to by the detachment of emitters from electrode surface as ZnTCPP barely soluble in pH 7.0 HEPES. Nevertheless, after taking resort to TOAB, intensive ECL emission with onset/peak potentials at −1.26/−1.55 V did occur on TOAB/ZnTCPP/GCE (Figure 3A, curve e), which approximate the position of Peak I. In this case, TOAB, as one kind of phase-transfer reagent, probably protected the transition states of ZnTCPP against the nucleophilic attack of water or H+ by exposing its hydrophobic aliphatic segment outwards.37 Given no luminescence out of mere TOAB (Figure 3A, inset c) as well as TOAB/TCPP (Figure 3A, inset d), the ECL radiation was thus accredited to ZnTCPP solely, while the intensity of its counterpart in oil phase became relatively weak as 75.03% of that of TOAB/ZnTCPP even at its 10-times amount (Figure 3A, curve a). This corroborates the reports that emulsifier and monomer would ameliorate the spatial relationship among metalloporphyrins on a large scale,15 rendering high luminous efficacy of ZnTCPP in the buffer. Moreover, because of the anionic conduction of TOAB,22,31 the ECL over-potential of ZnTCPP/SPE moved towards zero for nearly 150 mV, comparatively of rather less interference 13 ACS Paragon Plus Environment


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from electroactive coexistents than our previously devised zinc proto-porphyrin IX@laponite nanocomposite.20 Even more impressed, once O2 and H2O would completely compromise ECL, whatever it came from carbonyltetrakis(3-sulfonatomesityl)porphyrin|oxalate, ruthenium(II) 2,3,7,8,12,13,17,18-octaethyl-21H,23H-porphine|TPrA, platinum(II) octaethylporphyrin|TPrA or magnesium chlorophyllin.20,38 Now by using TOAB, these ECL chromophores of aprotic library could instead be well inherited and improved in common aqueous regimes. On the other hand, in O2-saturated solution, whose concentration quintuples the partial pressure of dissolved O2 at normal atmosphere, the ECL emission at the layer of TOAB/ZnTCPP enhanced fivefold accordingly (8005 vs. 1586 in arbitrary unit (a.u.)) under a alleviated PMT bias of −600 V (Figure 3A, curve f), which accompanied by a surge of oxygen-reduction current around −0.55 V (Figure 3B, curve b). Presumably due to the oxygen-triggered parallel catalysis,39 all ipc increased; while the negative shifts in both ECL and CV peaks could be justified by Nernstian equation with respect to adequate coreactants. As expected, the ECL of ZnTCPP diminished entirely in the absence of O2 (Figure 3A, curve g), which demonstrates that O2 affects the rate-determining step over this cathodic ECL mechanics. Apparently, it assumes to consist of two aspects: ZnTCPP and O2. In a nutshell, a new flexible ECL electrode of TOAB/ZnTCPP was established. Concerning that the aqueous solubility of air under the standard status is ~320 μM, the endogenous O2, as a ready-made coreactant of less than 60 μM, seems enough to energize an efficient unipolar ECL event of ZnTCPP, which is considered to be a potent and handy alternative in the cathodic route. 3.3. Interrogation on Electro-Photodynamic Origination. To illuminate the ECL derivation of ZnTCPP, first of all, the ECL spectra were profiled and powered by chronoamperometry at −1.6 V. In air-saturated aqueous medium, TOAB/ZnTCPP/GCE exhibited a conspicuous ECL phenomenon with a full-width at half maximum (fwhm) of 24 14 ACS Paragon Plus Environment

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nm and good monochromaticity at 634 nm (Figure 3C, black solid); while TOAB/TCPP/GCE could barely light up (Figure 3C, black dashed), TOAB/GCE either (data not shown). The most striking thing lies in that TOAB/ZnTCPP/GCE managed a considerable red emission without the help of excitation rays. In the meantime, by virtue of such substantial photon counting (> 8×105 a.u.) from ZnTCPP-based ECL in fluorimeter, there were reasons to believe that the emitting site could be eye-witnessed straightly through a camera lens, while elements of optical path are dispensable. Fulfilled and exemplified as Figure 3C inset, a greyish-white circular area with an obscure circumference, which was initially the TOAB/ZnTCPP-covered working surface of SPE, presents lively in a dark background (noise). This dramatic contrast in grayscale reflects the inherently superior visibility of ECL technology.2−4 Similar to the assessment of PL quantum yield, the ECL efficiency (ΦECL) of ZnTCPP|O2 system was determined to be 0.822, competent with that of ruthenium(II) tris(2,2′-bipyridyl) (Ru(bpy)32+)| tri-n-propylamine (TPrA) (ΦECL = 1.0),1 and undoubtedly prevailing over the performances of semiconductor nanocrystal family.40 Bearing resemblance in excitonic luminescence with the ECL technique, molecular spectroscopy was hyphenated with the aforementioned ECL spectra in the second place. As shown in Figure 3C, the optical absorption of TCPP underscores a sharp Soret band (λab)max at 419 nm tailing with serial Q-band wavelets (Figure 3C, blue dashed). In the presence of Zn2+ coordination, the nonsplitted Soret peak of ZnTCPP red-shifted to 428 nm (Figure 3C, blue solid), which was construed as no apparent J-aggregates in addition to the abatement of π-conjugation.36 This 9-nm discrepancy in the ground-state absorbance, e.g. the optimal PL exciting wavelength (λex)opt, further diverged the static fluorescent properties of TCPP and ZnTCPP. The latter have one narrow symmetric peak at 608 nm and a shoulder at 660 nm (Figure 3C, red solid), which contrarily grew into 650-nm major for TCPP alongside with 15 ACS Paragon Plus Environment


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lesser emission at 716 nm (Figure 3C, red dashed). An internal conversion (Soret → Q) must happen after the excitation as the emission maxima partially coincided with Q bands.36 Likewise, there are just too many vibrational modes in ZnTCPP to preclude its fine vibronic configuration, which means this PL survey scan at certain λex may not catch fully insight of the presented ECL in question. Yet by referring to the fundamentals of photodynamic therapy,21,31 this ″monochromatic″ ECL output is believed to be stemmed from a much simpler molecule in the same system. Being aware of that, one would come to realize the ordinary paramagnetic O2, with two unpaired electrons in its degenerate frontier orbitals (termed as 3O2 for a total spin-multiplicity of 3, 3Σg in Schonflis symbol), became the suspected ECL source behind the scene. It is well-known that singlet oxygen (1O2, 1Δg state) is the spin-paired product of 3O2 after its uptake of slight energy from electron donors.22 Afterwards, dimol 1O2 react mutually to release the triplet ground-state 3O2 concomitantly with the characteristic red chemiluminescence (CL, λem):31 1

O2 + 1O2 → 2(3O2) + hυ

(λem = 634 and 703 nm)

(5)

This inference is apparently rationalized by Figure 3A and 3B, where both ECL intensity and ipc escalated with the accumulation of O2, and was solidly elucidated by gradual depletion of 1O2 in the presence of 10 mM NaN3 (Figure 3D).42 Contradictorily, on such occasion, O2 would be taken as a coreactant for granted,40 while ZnTCPP was empirically thought to be the origin of ECL. To clarify the ambiguity of this aerobic pathway, several experiments, especially in situ EPR observation, were conducted. First and foremost, supposing that ZnTCPP was the exclusive photosource, other kinds of oxidants, for instance peroxysulfate (S2O82−) and the like, may be adapted to attain an ECL amplification of ZnTCPP, in a way like they promote the QDs-based ECL.40 Unexpectedly, 16 ACS Paragon Plus Environment

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unlike TCPP-assisted ECL of S2O82−,41 so much as 1.0 M S2O82− could not switch the emission on in deoxygenized buffer (Figure 4A, curve a). Also, following a conventional annihilation course via redox cycling in nitrogenous solution (like Figure 2A), still sees no sign of recovery (Figure 4A, curve b). Both jointly confirm that ZnTCPP lacks the self-luminous ability in ECL, yet somehow owns a special dependency upon O2. 1O2 can be mostly produced indirectly from aqueous reactions of superoxide anion (O2•−), the one-electron reduction product of molecular oxygen.24 As a tentative preliminary, 5 mM L-cysteine, as a scavenger to free superoxide (O2•−) and hydroxyl (OH•) radicals,42 was introduced into the air-saturated detection solution, which surprisingly begot a perfect quenching on the ECL emission of TOAB/ZnTCPP during continual potential sweep between 0 and −1.7 V (Figure 4A, curve c). Furthermore, 0.2 mg·mL−1 SOD, a specific catalyst for O2•− with a rate constant up to 109 M−1·s−1,43 was involved in the system, which dropped instantly to a 100% extinguished ECL response (Figure 4A, curve d). These illustrate that O2•− species as an intermediate of the dissolved oxygen participates in the whole progress. To prove the instantaneous formation of O2•− and its potential-modulated conversion to 1O2 at modified SPE, in situ EPR platform was constructed in tandem with potentiostat and circulated for five times. As a blank, the deaerated pH 7.0 HEPES containing 50 mM DMPO as the spin trap did not display any EPR traits (Figure 4B, curve a). Kept in dark place and applied O2-reduction potential (−0.6 V) upon TOAB/ZnTCPP/SPE in HEPES containing 60 μM O2 and 50 mM DMPO for a duration of 5 min, six hyperfine splitting peaks raised at 3446, 3462, 3471, 3477, 3486 and 3501 G (Figure 4B, curve b), which altogether stands for the emergence of DMPO−O2•− adduct,24,42 a strong evidence verifying the presence of O2•−. No other reactive oxygen species (ROS) were signified. Concurrently, replacing DMPO with TMP brought about none 1O2-entrapped signal - a group of three congruous Zeeman splits (3463, 17 ACS Paragon Plus Environment


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3480 and 3498 G) from tetramethylpiperidine-1-oxyl, unless at −1.2 V (Figure 4B, curve c), where the reduced Zn•+TCPP formed (Figure 2A, curve c) and the ECL initiated (Figure 3A, curve e). Its intensity lifted as the potential settled more negatively from −1.3 to −1.7 V, and rose to the strongest at −1.6 V (Figure 4B, curve d), neighboring the ECL peak potential. Coincidentally, when 430-nm laser fiber shone incessantly upon the power-off electrode, the EPR instrument also detected 1O2 out without any Gaussian displacement (Figure 4B, curve e), and the reading built up over the irradiation time. This measure was intended to simulate the photodynamic circumstance, in which ZnTCPP as a photo-sensitizer agent was affirmed to deliver ROS, matching Type I model in concepts of photodynamic therapy in clinics.31 Back to the ECL issue, 1O2 was believed to be created and regulated by further electro-reduction at rather negative potential. These EPR analyses substantiate that the ECL emission begins with the transformation of O2 into O2•− at TOAB/ZnTCPP/SPE: O2 + e− → O2•−

(6)

Although the involvement of 1O2 in ECL has been studied occasionally and arguably, such as electron-transfer annihilation reaction in nonaqueous media containing ferricenium cation and TPrAH•+,43 and intensification of ROS on the surface of indium tin oxide,44 1O2 generation in the cathodic electrochemical process of porphyrin with such luminescent phenomenon has never been reported explicitly so far. Taking consideration of the paralleled catalytic wave in Figure 3A inset, the electron in Eq.(6) was in fact retrieved from the negative-charged Zn•+/0TCPP in Eq.(3) and (4), the Electron Transfer of which could be expressed as: Zn•+/0TCPP + O2 → Zn2+/•+TCPP + O2•−

(7)

A more complicated kinetics might also be operative where 1O2 is produced when O2•− reacts backwards with the product (or reactant) of Eq.(4) (an E−C mode),39 e.g. Zn•+/0TCPP or 18 ACS Paragon Plus Environment

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even intermediate like Zn•+TCPP•−, since their electrode potentials are very close to each other: Zn2+/•+TCPP + O2•−  Zn•+/0TCPP + 1O2

(8)

Zn•+TCPP•− + O2•−  Zn•+TCPP2− + 1O2

(9)

Eq.(8) and (9) constituted the precursory reactions for Type I photodynamics that O2•− was oxidizes into 1O2,24,31 and resembled the so-called ″reductive-oxidation″ in coreactant-based courses.40 Obviously, the dissolved O2 can significantly promote electrochemical redox processes of chelated Zn and TCPP (Figure 3A, inset b). On the other side, being the most probable hopping as ligand-to-metal charge transfer (LMCT) during the intersystem crossing,20 an excited singlet state of ZnTCPP (1ZnTCPP*, S1) remained immediately following the oxidation-reduction of Eq.(7)−(9), and relaxed (3ZnTCPP*, T1). Excessive 3O2 as the energy acceptor would inevitably continue to collide with S1/T1, engaging in Energy Transfer: 1

ZnTCPP* (S1) + 3O2 (3Σg) → 3ZnTCPP* (T1) + 1O2 (1Δg)

(10)

3

ZnTCPP* (T1) + 3O2 (3Σg) → 1ZnTCPP (S0) + 1O2 (1Δg)

(11)

where 1ZnTCPP* returned to its ground singlet state (1ZnTCPP). This extrapolation was reasoned analogically by time-resolved PL spectroscopy on the score of its similarity with ECL in excitonic luminescence. Once pulsed at (λex)opt = 428 nm, the time-resolved PL plot of ZnTCPP in air-saturated HEPES (Figure 4C, curve a) decayed rather more promptly than that in N2 (Figure 4C, curve b). The attenuation could be well fitted exponentially into the polynomial: f(t) = A + B∙exp(−t/τ) with a 100% relativity, where the time constant τ for both situations were calculated to be 2.6 and 9.0 ns, respectively. This mono-exponential factor manifested that, for one thing, the radiative recombination of ZnTCPP passed by its excited singlet state (S1); for another, 3O2 as the energy receptor shortened notably the lifetime of S1. Thereafter, Eq.(10) and (11) evolved rapidly into Eq.(5), the key step along the route of Type II



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photodynamics:21 1

O2 (1Δg) + 1O2 (1Δg)  (1O2)2*

(1O2)2*  3O2 (3Σg) + 3O2 (3Σg) + һυ

(12) (em  634 nm)

(13)

In summary, a stepwise formulae set are described above. The overall elaboration could be briefed as a ZnTCPP-electrocatalyzed CL emission of O2, and can be termed electro-photodynamics, that are responsible for the multistate sensitization of ground-state oxygen. In that sense, a new gaseous ECL emitter is discovered, just right being the naturally ubiquitous oxygen on the earth. Allowing TOAB/ZnTCPP for an unusual ″solid coreactant″ or ″electro-photosensitizer″ to amplify signals, its operation mode was categorized to be disparate from classical ECL paradigms, otherwise sharing essential characteristics with the photochemistry of pigments. As a particular supplementary, the electrochemical responses of TOAB/ZnTCPP/GCE to the contents of dissolved O2 were depicted in Figure 4D. With the increasing O2 concentration, ipc augmented gradually and reached a plateau, which obeyed the Langmuir-type adsorption isotherm as shown in Figure 4D inset:39 [c]/ipc = [c]/(ipc)max + b−1/(ipc)max

(14)

where [c] represents the concentration of O2, b is defined as the apparent adsorption equilibrium constant, and (ipc)max maintains the ipc at maximal adsorption. As a result, the value b of O2 at TOAB/ZnTCPP/GCE was quantified to be 5.71×103 L·mol−1 (Figure 4D, inset a) nearly quadruples that at bare SPE (1.46×103 L·mol−1) (Figure 4D, inset b), which was perhaps rendered by the infiltration of O2 into multiple vacancies of TOAB with high specific area. The dissolved O2 could be replenished soon enough by pneumatic feed. In this regard, Eq.(7)−(10) are factually experiencing an adsorption-incurred catalysis of O2 at ZnTCPP, whose reduction


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rate was drastically accelerated, culminating in an extraordinarily intense ECL, visible to naked eyes. In short, TOAB not only offered ECL accessibility for the hydrophobic ZnTCPP in polar solvents but also enhanced its 1O2 quantum yield. Being a curiosity to weigh the 1O2 quantum yield at TOAB/ZnTCPP (ΦZnTCPP), the degradation of ADPA was tracked as soon as it trapped 1O2 generated periodically by the electric stimulus of TOAB/ZnTCPP and photo-excitation of Rose Bengal as the standard photosensitizer.31 The long-term absorptivity at 378 nm fell slowly in Figure 4E upper, and the deterioration of ADPA by Rose Bengal was far more prominent than that by ZnTCPP, as ΦZnTCPP was statistically to be 0.27. The 634-nm 1O2 emission induced by ZnTCPP-aroused ECL and the PL of Rose Bengal in CH2Cl2/TBAP were combined in Figure 4E lower, and ΦZnTCPP was formulized to be 0.24. Both numerical values echo well reciprocally. To the best of our knowledge, this is the first 1O2-generating efficiency ever reported in the ECL field, unanticipatedly surpassing some state-of-the-art photo medicine.24 3.4. Optimization of Parameters. Motivated by the ″turn-on″ effect of dissolved O2 on the ECL at TOAB/ZnTCPP/SPE, the brightness of which was so eye-catching that one could see it through camera, a feasible strategy for visual quantification of trace aqueous oxygen came into being. Ahead of zooming in, focusing lens and taking shots, several experimental prerequisites should be optimized including the electrolytes, the solution pH, the proportion of ZnTCPP to TOAB, the constant electrolyzing voltage, and the duration of consecutive exposure. Firstly, to run this aerial ECL system at its best, the liquid environment must be selected among a number of available components, such as carbonate, phosphate, borate, acetate, tris(hydroxymethyl)aminomethanebase (Tris), and HEPES. Every saline holds a distinctive buffer capacity. As shown in Figure 5A and Figure 5A inset, the normalized ECL intensity 21 ACS Paragon Plus Environment


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differs from one salt to another, and climbs atop by a narrow margin in pH 7.0 HEPES, in spite of a bit more negative potential. Apart from that, a climax at −1.33 V with onset as low as −0.94 V could be acquired in pH 9.0 HCO3−/CO32− (Figure 5A, curve c), which is obviously inappropriate for mimicking physiological conditions. Generally speaking, owing to the isolation of water from ZnTCPP by TOAB, this ECL system behaves quite tolerant of polar protonic solvents, and better than ECL of QDs with volatile surface-states vulnerable to solvation.40 At the same time, the luminance turned into be less susceptible to the perturbation of acidity, keeping roughly in a line as the pH value alternates from 6.0 to 9.5 (Figure 5B and Figure 5B inset), which succeeded in securing the active intermediates. Taking biocompatibility into account, a 10 mM pH 7.0 HEPES was finally chosen as the detection solution for the following visualization and calibration. Secondly, the intake of ZnTCPP among the intermolecular gap of TOAB was tuned on the basis of porphyrin mass: 0.5~3.0 mg (0.6~3.6 mM) ZnTCPP as the univariate, mixed with six equivalent TOAB (10.9 mg, 20 mM) and embodies as Figure 5C and Figure 5C inset, of which 2.0 mg (2.4 mM) ZnTCPP overwhelms all else. Beyond this critical point, the leakage of overloaded ZnTCPP might aggravate desorption of TOAB from the SPE. Conversely, the underloaded ZnTCPP could never meet its saturation in a huge composition of TOAB, which definitely impacts on the ECL output. Thirdly, in the light of that intensity altered with cyclic scan (Figure 3A), to guarantee most legible pictures, observations of single facula were undertaken in a reiterative manner between −1.2 and −1.7 V. Aligned in a dark-light gradient strip, the colors of distinguishable circles faded up and down coherent to the saddle-shaped ECL profile (Figure 5D). The human eye is able to capture such changes even at weak intensity. The brightest ray emerges at −1.6 V, which agrees with the peaking potential in Figure 3, hence wades handpicked to evoke the 22 ACS Paragon Plus Environment

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imaging. Browsing through Figure 5D lower, three interesting subtleties could be perceived: (1) Discriminated from homogenous CL and PL imaging, which always project uniform illumination,45 ECL imagery prefers an irregular light distribution in close proximity to the electrode; (2) There are sparsely scattered black regions like sunspots in faculae, corresponding to less reactive sites on modified SPE; and (3) The periphery whitens in advance of the interior, possibly because the molecular O2 was inclined to the perimeter where TOAB/ZnTCPP spread relatively thin or the solution wetted easily at this three-phase (SPE|TOAB/ZnTCPP|HEPES) boundary. Virtually, this array of samples at varied stationary potentials comprises a vivid trajectory of CV-steered ECL, perfectly explaining its immanent spatial and temporal disparities. The so-called ″time-dependent″ has another meaning, since CCD camera requires long exposure to secure quality images for a sensitive assay.45 As time lapsed, the luminosity of spot ascended very quickly and accessed to its acme within 60 s (Figure 5E lower). Therefore, an exposure time of 1 min was used for dynamic integration to visualize ECL, which could circumvent the possibility of over-exposure and assure a high throughput. Additionally, the ECL of TOAB/ZnTCPP/GCE also exhibits reliability, anti-photo-bleaching and resilience against prolonged cyclic scans with a relative standard deviation (RSD) in selected peak intensities of 1.6% (Figure 5E inset). According to Kumar and Bard,44 electrode defouling was necessary to avoid the passivation by byproducts and to reproduce the ECL signal. However, it is not an issue in this work, because images sampled at intervals were retained at an invariable gray-level after successive electrolysis for up to 30 min (Figure 5E), and could endure an elongated time-span further, which should be counted as an advantage over the decay of pure CL.45 Eventually, a brand-new setup of ECL has been invented and was ready for pertinent exercises. 23 ACS Paragon Plus Environment


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3.5. Chip-Based ECL Visualization of O2 and Analytical Performance. Under the optimum conditions and at 20 °C, 1.02×105 Pa, the optical density was proportional to the increasing concentration of dissolved oxygen (Figure 6). The calibration function displayed a good linearity between the mean pixel and the concentration of O2 in a detection range from (approximate molarity) 6.3 to 69.3 μM (Figure 6 inset), with a correlation coefficient (R2) of 0.999. The lower limit of detection (LOD) at a signal-to-noise ratio of 3 (n = 3) was 4.2 μM, which was at least 10 times lower than results from the microrespirometer,46 and main sorts of electrocatalysis toward oxygen reduction,47 and rivals against the commercialized Clark-type oxygen electrode (e.g., ~100 μmol·mL−1 out of Chlorolab-2TM Liquid-Phase Oxygen Measurement System, and OxythermTM Liquid-Phase Photosynthesis & Respiration Measurement);48 while the upper one exceeds the critical saturation of dissolved O2 (i.e., oxygen limitation chemostat) at ~0 °C, 105 Pa (14.64 mg·L−1, 45.7 μM).48 More importantly, this lab-on-chip based imaging prototype was unsophisticated, disposable and visually sensible. As for peer competitors, our system owns as much as quintuple sensitivity to a reversible gas sensor based on ECL redox of Ru(bpy)32+ (LOD: 20% (v/v) of air, i.e., 360 μM);49 doubles that of a molecular oxygen bipolar detector by ferrocenemethanol-inhibited ECL of Ru(bpy)32+ (LOD: 300 ppb, 9.38 μM);50 and even takes the lead against the

plausible

luminol−O2−scavengers (glucose oxidase, superoxide dismutase) system (LOD: 55 μM).51 To evaluate the anti-interfering ability of our proposed system, the influences of common 14 anions and 17 cations on the ECL imaging were examined. Either anionic (Figure 7A) or cationic (Figure 7B) hydrated candidates only incited negligible ECL fluctuation upon TOAB/ZnTCPP/SPE even at a rather high concentration of 1.0 mM, suggesting excellent ionic resistance, thus would not deprive 1O2 of its functionality as ECL origination. In practice, the coverage of TOAB serves as the thin film (thickness: 15~20 μm) of polyethylene or 24 ACS Paragon Plus Environment

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polytetrafluoroethylene (Teflon®), preventing electrolytic reactions and allowing only the dissolved O2 to penetrate through. The intra-assay and inter-assay errors of ECL visualization were examined by detecting 160 μM O2. The RSD for fifty measurements with the same SPE was 1.2%, while that for ten parallel measurements with thirty separate pieces of SPE was 3.0%, indicating satisfactory precision and acceptable reproducibility. By consulting results of commercial Oxygraph Plus System (Hansatech Instruments, UK), the proposed method showcased an accurate determination of dissolved oxygen (Table 1), the feasibility of which would ultimately aim at tangible researches on Biochemical Oxygen Demand (BOD), Chemical Oxygen Demand (COD), respiratory/photosynthetic metabolism, oxidase activity, etc.

4. CONCLUSIONS In this work, an electro-photodynamic imaging of 1O2 came true on TOAB/ZnTCPP. Spectroscopies and in situ EPR testified the reality of ECL from 1O2 and how to manipulate it potentiometrically; while the cathodic reduction of ZnTCPP associated its reactive intermediates with O2•−/3O2 precursors in ways of electron and energy transfer. The utilization of TOAB not only solved the difficulty in aqueous vitality of luminophores, but also endowed them with regular filming pattern, swift signal transduction, and succedent high

1

O2

productivity. Such a trinity of ZnTCPP, TOAB and O2 casts cohesively a green, economic and concise ECL systematic innovation. Further prototyping and parameterization successfully visualized 1O2 on-chip at sub-nanomolar level with veracity and repeatability. Therefore, zinc porphyrin, as a promising ECL sensitizer, infuses fresh blood into traditional ECL technologies by implanting the omnipresent airborne emitter 1O2 inside the connotation of ECL. Last but not 25 ACS Paragon Plus Environment


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the least, taking account into the state-of-the-art progress in in vitro/in vivo monitoring of hydroxyl radicals,52 inspection concerning the adaptability of our system to such target is currently underway.

■ ACKNOWLEDGMENTS This research was supported by National Natural Science Foundation of China (Grant No. 21305067 and 61371039), Natural Science Foundation of Jiangsu Province (BK20130754), Ph.D. Fund of Ministry Of Education for Young Teachers (0133219120019), the Fundamental Research Funds for the Central Universities (30916011204), Qing Lan Project of Jiangsu Province, and ″A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)″.


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Table 1. Assay Results of Water Samples Using the Proposed and the Reference Methods Sample No.

1a

1b

1c

2

3

4

Proposed Method

(mg·mL−1)

9.28

13.08

7.04

9.25

8.98

9.48

Reference Method

(mg·mL−1)

9.16

12.84

6.85

8.90

8.73

9.27

Relative Error

(%)

1.3

1.9

2.8

3.9

2.9

2.3

a, b, and c

This sample was pretreated at 20, 5, and 35 °C (±0.2 °C), respectively; others were at 20±0.2 °C.


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FIGURE CAPTIONS: Scheme 1. (A) General principle of the microchip-based electro-photodynamic visualization, and (B) Schematic mechanisms for electro-photodynamics of 1O2 at TOAB/ZnTCPP/SPE. Figure 1. FESEM images of (A) ZnTCPP/SPE and (B) TOAB/ZnTCPP/SPE. Inset B: XRD pattern of TOAB/ZnTCPP. Figure 2. (A) CVs of TOAB/GCE (a), ZnTCPP/GCE (b), TOAB/ZnTCPP/GCE (c), and TOAB/TCPP/GCE (d) in N2-saturated HEPES. Inset A: Selected range magnified. (B) SWVs of TOAB/ZnTCPP/GCE (a) and TOAB/Zn/GCE (b) in the N2-saturated HEPES (Frequency: 15 Hz, Potential step: 4 mV, Pulse amplitude: 25 mV). Inset B: Plots of ipc (I, II and III) vs. the square root of scan rate. Figure 3. (A) ECL-potential curves of ZnTCPP/GCE in air-saturated DCM/TBAP (a) and HEPES (b); TOAB/GCE (c) and TOAB/TCPP/GCE (d) in air-saturated HEPES; and TOAB/ZnTCPP/GCE in N2 (e), air (f) and O2 (g) saturated HEPES. Inset A: Magnified (b), (c) and (d). (B) CVs of TOAB/ZnTCPP (a)(b) or TOAB/TCPP (c)(d) modified GCE in N2 (a)(c) and O2 (b)(d) saturated HEPES. (C) ECL (black), PL emission (red, λex = 430 nm) and UV-Vis absorption (blue) spectra of TOAB/ ZnTCPP/GCE (solid) and TOAB/TCPP/GCE (dashed). Magenta dot: TOAB. Inset C: Typical TOAB/ ZnTCPP/SPE with (upper) and without (lower) ECL in CCD. (D) Cyclic ECL responses between 0 and −1.7 V of TOAB/ZnTCPP/GCE in air-saturated HEPES containing 10 mM NaN3. Figure 4. (A) ECL-potential curves of TOAB/ZnTCPP/GCE on different conditions: in N2-saturated HEPES (left) + 1.0 M S2O82− (right) (a), by negative-positive potential cycles as Figure 2A, curve c (b), in O2-saturated HEPES (left) + 5 mM L-cysteine (right) (c), and in 35 ACS Paragon Plus Environment


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O2-saturated HEPES (left) + 0.2 mg·mL−1 SOD (right) (d). (B) Accumulated EPR signals of TOAB/ZnTCPP/SPE in deaerated HEPES+50 mM DMPO (a); (a)+60 mM O2 recorded at −0.6 V for 300 s (b); deaerated HEPES+50 mM TMP+60 mM O2 at −1.2 V (c), and −1.6, −1.7, −1.5, −1.4 and −1.3 V (from top to bottom) (d) for 300 s; and (c) upon 430-nm irradiation for 5 min without electricity (e). (C) Time-resolved PL spectra of ZnTCPP in air (a) and N2 (b) saturated HEPES ((λex)opt = 428 nm). Inset C: Corresponding static spectra. (D) CVs of TOAB/ZnTCPP/GCE at increasing concentrations of O2 (from top to bottom) in N2-saturated HEPES at 300 K. Inset D: O2 adsorption isotherms at TOAB/ZnTCPP (a) and TOAB (b) modified GCEs. (E) Upper: The normalized absorbance of ADPA at 378 nm as a function of irradiation time in the presence of TOAB/ZnTCPP (a) and Rose Bengal (b); and Lower: The 1

O2 emissions at 634 nm induced by ECL of TOAB/ZnTCPP (a) and PL of Rose Bengal (λex =

430 nm) (b) in DCM/TBAP. Figure 5. Effects of (A) the electrolytes and pHs (from f (top) to a (bottom): HEPES 7.0, Tris 8.0, acetate 6.3, borate 8.9, phosphate 7.4, carbonate 9.0), (B) the solution pH: 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, (C) the proportion of ZnTCPP (0.6, 1.2, 1.8, 2.4, 3.0, 3.6 mM) to TOAB (20 mM), (D) the constant electrolyzing voltage (forwards: −1.2, −1.3, −1.4, −1.5, −1.6, −1.7 V, and backwards), and (E) the duration of consecutive exposure (10, 20, 30, 40, 50 s, and 1, 5, 15, 30 min) on the insetting ECL images. Inset E: ECL emission cycles of TOAB/ZnTCPP/SPE in air-saturated HEPES. Figure 6. The ECL images of O2 at 6.3, 15.2, 27.0, 35.3, 44.6, 54.4 and 69.3 μM; and the corresponding calibration curve. Figure 7. Normalized ECL intensities of TOAB/ZnTCPP/SPE in air-saturated HEPES with 1.0 mM manifold (A) anions and (B) cations. 36 ACS Paragon Plus Environment

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Scheme 1



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Figure 1


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Figure 2



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Figure 3


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Figure 4



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Figure 5


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Figure 6



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Figure 7


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Table of Contents Graphic


Electro-Photodynamic Visualization of Singlet Oxygen Induced by Zinc

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