Steady-State Electrochemiluminescence at Single Semiconductive

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Steady-state Electrochemiluminescence at Single Semiconductive Titanium Dioxide Nanoparticles for Local Sensing of Single Cells Chen Cui, Ying Chen, Dechen Jiang, Hong-Yuan Chen, Jianrong Zhang, and Jun-Jie Zhu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04778 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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Analytical Chemistry

Steady-state Electrochemiluminescence at Single Semiconductive Titanium Dioxide Nanoparticles for Local Sensing of Single Cells Chen Cui, Ying Chen, Dechen Jiang*, Hong-Yuan Chen, Jianrong Zhang*, Jun-Jie Zhu*

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, 210093, China

Corresponding Authors: Dechen Jiang ([email protected]); Jianrong Zhang ([email protected]); Jun-Jie Zhu ([email protected]).

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ABSTRACT: Obtaining steady-state bright electrochemiluminescence (ECL) at single nanoparticles is crucial but challenging for the realization of the single-nanoparticle electrochemical sensing of single cells.

In this

work, steady-state bright ECL is implemented at single semiconductive titanium dioxide (TiO2) nanoparticles for sensing the local efflux from single living cells.

Oxygen vacancies on the surface of rutile TiO2

nanoparticles have a high affinity for hydrogen peroxides that are not easily passivated upon exposure to voltage.

Therefore, the steady-state adsorption of hydrogen peroxide by the TiO2 nanoparticle surface

permits the continuous electrochemical generation of superoxide and hydroxyl radicals by electrons and surface-trapped holes at the nanoparticles, resulting in constant ECL under physiological conditions.

This

steady-state luminescence during continuous imaging is correlated with the concentration of hydrogen peroxide, leading to the local ECL visualization of hydrogen peroxide efflux from single cells.

The

successful local ECL imaging demonstrated herein provides an unprecedented approach to enable subcellular electroanalysis using single nanoparticles.


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Introduction Rapid developments in electrochemistry using single nanoparticles have spurred many exciting advances, including a better understanding of electrocatalytic mechanisms, elucidation of the particle structure−function relationship of nanoparticles, and construction of ultrasensitive sensors.1-6

However, currently, separation of

the signal at an individual nanoparticle from surrounding nanoparticles and/or environment remains challenging.

As a result, it is difficult to obtain spatially and/or temporally resolved information on

heterogeneous processes occurring on specific nanoparticles.

The combination of single-nanoparticle

electrochemistry with optical techniques offers promising high-throughput and submicrometer spatial resolution for the in situ imaging of local electrochemistry or nanoelectrochemistry without complicated microfabrication techniques.7-11

However, applying single-nanoparticle electrochemistry for the label-free

study of molecular distribution in single living cells has not yet been realized.

Accordingly, the

abovementioned challenge concerning the spatial/temporal analysis using single nanoparticles has not been fully solved.

Electrochemiluminescence (ECL) occurs when light is emitted from an electronically excited

intermediate during an electrochemical reaction that possesses a near-zero background readout and exhibits a high sensitivity to biomolecules.12-15

Single-nanoparticle ECL has thus become a highly promising tool in the

visual investigation of local biological events. Bard’s group has conducted pioneering work on single-nanoparticle ECL using sub-25-nm single immobilized polymer nanoparticles.9

Their results have provided dynamic information on heterogeneous

electron-transfer kinetics at the single-nanoparticle level.

The further application of novel metal

nanoparticles has revealed much stronger ECL, and this research has led to the visualization of ECL at single nanoparticles with microscopy.

The intensity of ECL is primarily controlled by the local chemical and

charge-transfer environment surrounding the nanoparticles.16

Because of the passivation of the metal surface

when under voltage, the ECL of metal nanoparticles quickly vanishes.

Both nanoparticles with a protective

layer and novel Au-Pt Janus nanoparticles have been reported to enhance local redox reactions and maintain the electrochemical activity of the particles involved.17,18

To facilitate the visualization of ECL at the

single-nanoparticle level, an alkaline solution and a special surface treatment for the nanoparticles have been used to increase the signal-to-noise ratio.

Despite implementing these improvements, metallic surface

passivation continues to be inevitable and poses a challenge to obtain stable ECL intensity.

Additionally, the

use of biologically incompatible solvents restricts the application of single-nanoparticle ECL for biological analyses.

Therefore, robust nanoparticles with stable and strong ECL under physiological conditions are

essential for the single-nanoparticle electroanalysis of individual cells with high spatial and temporal resolution. In this work, steady-state bright ECL from luminol and hydrogen peroxide is first observed from semiconductive titanium dioxide (TiO2) nanoparticles on an indium-tin oxide (ITO) surface in a physiological solution (Figure 1).

Sufficient oxygen vacancies located on the surfaces of TiO2 nanoparticles are known to

have a high affinity for hydrogen peroxide.

Additionally, TiO2 can accelerate electron transfer.


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Subsequently, aqueous hydrogen peroxide and dissolved oxygen can be electrochemically reduced by TiO2 nanoparticles, generating superoxide radicals (O2•-) and hydroxyl radicals (OH•).19,20

Once the voltage has

changed the oxidative potential of the luminol, aqueous luminol can be oxidized into luminol intermediates at the ITO and TiO2 surfaces, which react with radicals to emit luminescence (λ ~ 420 nm).

Furthermore, TiO2

nanoparticles can absorb luminescence, which results in the separation of electrons and holes.19-22

The

surface-trapped holes can then oxidize hydrogen peroxide to produce superoxide radicals, which produce more luminescence.23,24

Eventually, luminescence produced by the nanoparticles is sufficiently strong to

distinguish these nanoparticles from the surrounding ITO surface.

Compared with easily passivated novel

metal surfaces, oxygen vacancies in TiO2 crystals are not easily passivated under regular voltage, which allows the surface conditions needed for ECL generation to be maintained.25,26

Therefore, the use of steady-state

bright ECL of semiconductive TiO2 nanoparticles for the analysis of local hydrogen peroxide efflux from single living cells is a reasonable proposal.

Spatially and temporally resolved single-nanoparticle ECL

should provide more data from multiple cellular regions at one cell than classic electroanalysis using single microelectrode.27,28 Experimental details Materials

and

Reagents.

TiO2

powder

8-Amino-5-chloro-7-phenylpyrido[3,4-d]

(P25)

was

purchased

pyridazine-1,4(2H,3H)-dione

from

Degussa

(L012,

Co.

(Germany).

molecular

C13H9ClN4NaO2) was obtained from Wako Chemical USA, Inc. (Richmond, VA, USA).

formula:

Sylgard 184

(including PDMS monomer and curing agent) was obtained from Dow Corning (Midland, MI, USA).

HeLa

cells were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences of Chinese Academy of Science (Shanghai, China).

HeLa cells were seeded in high-glucose

medium supplemented with 10% fetal bovine serum and 1% antibiotics (penicillin/streptomycin) at 37 °C under a humidiﬁed atmosphere containing 5% CO2. Synthesis and Characterization of TiO2 Nanoparticles. The TiO2 nanoparticles were synthesized by a modified hydrothermal growth method described in the literature.29 mL of a 15-M aqueous NaOH solution.

Briefly, 2.5 g of P25 powder was mixed with 40

To obtain solution uniformity, the mixed solution was sonicated for

30 min; carefully transferred into a clean, Teflon-lined digestion vessel; and heated at 180 °C for 48 h.

The

white product formed at the bottom of the Teflon vessel was obtained by removing the supernatant. Subsequently, the product was immersed in 0.5 M HCl for 6 h and washed several times with water until a neutral solution was obtained. product.

The TiO2 nanoparticles were dried under vacuum, resulting in the dry, final

Nanoparticles were produced at different temperatures, and nanowires were heated in a tube furnace

for 2 h. Luminescence Imaging. Images were obtained with a water objective (×40, Olympus, Japan) and an electron-multiplying CCD (EM CCD) camera (Evolve, Photometrics, Tucson, AZ).

TiO2 nanoparticles were

drop-casted onto ITO slides to produce a working electrode, and platinum (Pt) and Ag/AgCl wires were used


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for auxiliary and reference electrodes, respectively.

A switching of the voltage between 1 V (2 s) and -1 V

(0.5 s) was continuously applied to the ITO electrode by a voltage generator (DG 1021, Rigol, China).

The

solution for luminescence imaging was 10 mM PBS (pH 7.4) with 200 μM L012. Results and Discussion. Electrochemistry and ECL of TiO2 nanoparticles. Single anatase TiO2 nanoparticles were synthesized using a hydrothermal treatment and were continuously calcinated at 700 °C for 2 h. These reactions were confirmed by X-ray diffraction (XRD) data (Figure S1, supporting information).

Scanning electron microscopy (SEM) and

atomic force microscopy (AFM) images (Figure S2, supporting information) display the dimensions of the nanoparticles (~ 10 µm in length, ~ 500 nm in width and ~ 130 nm in height).

Increasing the calcination

temperature resulted in the transformation from the anatase phase to the rutile phase.

After calcination at

1000 °C for 2 h, pure rutile TiO2 nanoparticles were formed, as confirmed by the XRD spectrum (Figure S1, supporting information).

The nanoparticle dimensions decreased overall to ~ 2 µm in length, ~ 500 nm in

width, and ~ 40 nm in height (Figure S2, supporting information). The nanoparticles were drop-casted onto ITO slides that were then immersed in a physiological phosphate buffer saline (PBS) solution (10 mM, pH 7.4).

Cyclic voltammetry (CV) and ECL of L012 (a luminol analog

with higher luminescence efficiency) and hydrogen peroxide were used to analyze bare ITO surfaces and TiO2 nanoparticle-coated ITO surfaces in PBS (Figure S3, supporting information).

The synchronous increases in

the anodic current and luminescence intensity under positive voltage were indicative of the oxidation of L012 on the electrode.

The larger anodic current observed for the TiO2-coated ITO electrode might be ascribed to

faster oxidation rate of L012 at the TiO2 surface, which increased the luminescence intensity.

When the

voltage became more negative, an increase in the cathodic current was observed in the presence of TiO2 nanoparticles.

This enhancement supports the accelerated reduction of hydrogen peroxide at the TiO2

surface, which catalyzed the oxidation of L012, resulting in higher luminescence.

Moreover, the

photocurrent produced by the TiO2-coated ITO electrode (Figure S4, supporting information) suggests the separation of electrons and holes on the TiO2 surface, which most likely induced the higher luminescence observed from the nanoparticles. ECL images of single TiO2 nanoparticles. different phases on ITO slides.

Figure 2 A–C shows bright-field images of nanoparticles with

Compared with the luminescence from the ITO slide, nanoparticles with

anatase, anatase/rutile, and rutile phases exhibited higher luminescence intensities (Figure 2 D–F) after the continuous application of voltages between 1 and -1 V.

The luminescence distinction of the particles

permitted the visualization of ECL for single TiO2 nanoparticles on ITO slides without any surface treatment. The intensities produced by TiO2 nanoparticles decreased gradually when the positive voltage decreased from 1 to 0.7 V (Figure S5, supporting information), which corresponded to the decreased oxidation of L012 at a lower positive voltage.

In addition, when a fixed voltage of 1 V was used to induce the oxidation of L012,

switching the voltage to a less negative value led to the generation of a lower number of superoxide radicals.


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As expected, the weaker luminescence produced by TiO2 nanoparticles demonstrates that superoxide radicals play an important role in ECL emission from TiO2 nanoparticles.

The diffusion of these radicals during ECL

process was characterized to be ~ 2 µm30, and thus, the spatial resolution of this current ECL image is restricted in 2 µm. The luminescence intensity across individual rutile and anatase nanoparticles was plotted and is shown in Fig 2G and H.

The peak intensities for the rutile, rutile/anatase, and anatase phases were 212 ± 62, 135 ± 38

and 125 ± 18 a.u., respectively.

The dependence of luminescence on the particle phase indicates oxygen

vacancies are involved in luminescence generation.

Numerous studies have confirmed the presence of

oxygen vacancies on the surface of rutile TiO2 nanoparticles and in the bulk of anatase TiO2 nanoparticles.31,32 Accordingly, a large number of oxygen vacancies on the surface of rutile TiO2 nanoparticles could lead to the sufficient adsorption of hydrogen peroxide required for subsequent electrochemical conversion, which could produce enhanced luminescence.

Additionally, variations in the microstructure and size of nanoparticles

produce differences in the density of oxygen vacancies, the separation efficiency of electrons and holes, and the catalytic efficiency, all contributors to high heterogeneity in luminescence intensity, which was observed in this study (Figure 2G and H). As mentioned above, the dissolved oxygen in the surrounding solution could be electrochemically reduced at the surface of TiO2 particles and, thus, could be producing an inflated ECL intensity.33-35

To investigate

this possibility, the ECL of individual rutile TiO2 nanoparticles was imaged in the absence of hydrogen peroxide.

Compared with the luminescence observed in the presence of 1 mM hydrogen peroxide, the

nanoparticles alone produced a luminescence intensity of 36.6 ± 1.8% (Figure S6, supporting information), exhibiting the minor contribution that oxygen reduction contributes to the total ECL intensity.

Removing the

dissolved oxygen in the buffer decreased the ECL intensities produced by these nanoparticles, indicating dissolved oxygen contributes to luminescence during the studied electrochemical conversion.

In summary,

the ECL produced by TiO2 nanoparticles originates from the electrochemical conversion of hydrogen peroxide and dissolved oxygen in the surrounding solution. Because concentrated hydrogen peroxide generates a larger amount of superoxide via electrochemical oxidation/reduction, a relationship between the luminescence from the nanoparticles and hydrogen peroxide concentration was also established.

Figure 3A–E presents bright-field and luminescence images of rutile

TiO2 nanoparticles in 10 mM PBS containing 0, 0.1, 0.5, or 1 mM hydrogen peroxide.

To avoid the effects of

the nanoparticle dimensions on ECL intensity, the background luminescence of the nanoparticles in PBS was normalized to a value of 1.0.

As shown in Figure 3F, the normalized luminescence (or luminescence ratio)

from four nanoparticles shows a positive correlation with hydrogen peroxide concentration.

The correlation

is not linear, which might be caused by the escape of the generated radicals at high concentration of hydrogen peroxide.

The relative standard deviations of these luminescence ratios at each concentration were calculated

to be less than 5.2%.

This consistent luminescent response for multiple nanoparticles suggests the feasibility

of using TiO2 nanoparticles for the local ECL analysis of hydrogen peroxide.


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The key to developing a method for single-nanoparticle ECL analysis is obtaining steady-state luminescence from nanoparticles during continuous imaging that allows for the comparison of intensities at different analyte concentrations.

In this experiment, 180 luminescence images from individual rutile TiO2

nanoparticles were recorded continuously over a 90-min period.

During imaging, aqueous L012 and

hydrogen peroxide were partially depleted near the nanoparticle and ITO surfaces.

To replenish these

species, a restoration time of 20 s (in which no application of voltage occurred) was added between consecutive images.

To further minimize the effects from the fluctuating concentrations of L012 and

hydrogen peroxide, the ratios of the luminescence from the nanoparticles and the surrounding ITO regions were calculated to characterize ECL stability.

The average luminescence ratios from three nanoparticles with

similar dimensions and luminescence are presented in Figure 3G.

The relative standard deviation of these

intensities from 180 images is 2.6%, revealing the steady-state luminescence from rutile TiO2 nanoparticles. ECL sensing of local efflux of hydrogen peroxide from single cells.

Single rutile TiO2 nanoparticles on ITO

slides were used to sense the local efflux of hydrogen peroxide from single living cells.

Cells were randomly

cultured on ITO slides, and cells located on top of multiple nanoparticles were chosen for this experiment. The representative bright-field and ECL images of these cells are shown in Figure 4A and B. from bare ITO regions was ascribed to the electrochemical oxidation of L012. cells, cellular membrane ruffling was observed.

Luminescence

In regions containing adherent

This membrane ruffling creates small spaces between the

lower surface of the cells and the ITO slide, thus allowing these spaces to retain some amount of L012.30 During the ECL process, the relatively slow diffusion of L012 from the bulk solution to these small spaces was unable to replenish the consumed L012. when imaging ITO slides.

As a result, cellular regions with a low ECL intensity appeared dark

However, TiO2 nanoparticles located in these small spaces emitted high

luminescence because of the presence of L012 and dissolved oxygen; thus, the TiO2 nanoparticles appeared bright in this dark region. Upon stimulating cells using phorbol myristate acetate (PMA), the released hydrogen peroxide reacted with TiO2 nanoparticles when under voltage, inducing higher ECL than when no PMA was used (Figure 4C).36,37 Differences in the luminescence shown in Figure 4B and C were plotted and are shown in Figure 4D.

To

exclude the possible contribution of a false luminescence increase by cellular motion or changes in cellular morphology, the luminescence from nanoparticles under non-stimulated cells was continuously recorded over a 2-min period. information).

No obvious change in the luminescence intensity was observed (Figure S7, supporting The experimental results suggest the higher luminescence from stimulated cells originated

from the efflux of hydrogen peroxide at the nanoparticle–cell contact regions and oxygen/L012 intermediates generated near these regions.

This local observation using the scattered nanoparticles avoids the intermediate

cross-talking produced in classic planar ECL analysis and, thus, may provide more accurate information about local hydrogen peroxide efflux. To determine if the luminescence from TiO2 nanoparticles is stable throughout the experimental period, temporal information on the efflux of hydrogen peroxide after stimulation was obtained by continuously


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recording the luminescence intensity produced by nanoparticles underneath a single cell (Figure 4E).

To

minimize the effects of luminescence efficiency from nanoparticles on local efflux characterization, the luminescence ratio of the nanoparticles before and after stimulation was determined.

Similar to the

microelectrochemical analysis results,27,28 bursts of luminescence were observed from all three of the nanoparticles studied.

This phenomenon was ascribed to the instant efflux of hydrogen peroxide from cells.

The varying fluctuation of the luminescence ratio obtained for these nanoparticles could indicate varying hydrogen peroxide efflux at different cellular regions.

The peak luminescence ratios from 12 nanoparticles

beneath five cells are listed in Figure 4F, and large deviations in the luminescence ratios of these nanoparticles are obvious.

Because the nanoparticles were located randomly beneath cells, the scatter plot indicates the

efflux of hydrogen peroxide at different cell locations was heterogeneous. A calibration curve was established to quantify the efflux of hydrogen peroxide by injecting hydrogen peroxide near the cells and recording the luminescence produced from the nanoparticles.

During this process,

hydrogen peroxide diffused beneath the cells and generated luminescence upon reaction with L012.

The

calibration curve (Figure S8, supporting information) was used to estimate the maximum efflux of hydrogen peroxide, which was ~ 100 µM.

Notably, although only a micromolar concentration of aqueous hydrogen

peroxide was injected near the cells, its introduction might have induced oxygen stress inside the cells.

As a

result, additional hydrogen peroxide release might have occurred, resulting in error in the quantification of the efflux of hydrogen peroxide.

Moreover, due to the missing information concerning the volume of the spaces

underneath the cells caused by cellular membrane ruffling, the released amount of hydrogen peroxide from the cells could not be determined. Conclusions. The steady-state bright ECL of single semiconductive TiO2 nanoparticles was recorded to demonstrate local electrochemical sensing of cellular efflux for the first time.

The stable luminescence of the

nanoparticles enabled the development of single-nanoparticle electroanalysis at the single-cell level.

The

subsequent development of single-nanoparticle ECL for single-cell analysis will requires quantification of cellular efflux. Unlike the current strategy using aqueous luminol, luminol could be immobilized to the nanoparticles such that the observed luminescence is indicative of the amount of cellular efflux.

In addition,

further studies could involve loading multiple oxidases onto nanoparticles, which would permit the sensing of more molecules in the plasma membrane or the cellular efflux.

Moreover, the ECL intensity at single

nanoparticles should be continuously enhanced so that the temporal resolution during the imaging could be improved to obtain more biological information about the cellular activity. Acknowledgement. We would like to thank the National Natural Science Foundation of China (Nos. 21327902, 21335004, and 21427807) for their support.


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Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. SEM, XRD, and additional ECL images

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Figures and Captions. Figure 1. Schematic process of the ECL sensing of single TiO2 nanoparticles for visualizing local hydrogen peroxide efflux from single cells. Figure 2. Bright-field and ECL images of single TiO2 nanoparticles. (A–C) TiO2 nanoparticles annealed at 700, 900, and 1000 °C; (D–F) ECL images of TiO2 nanoparticles corresponding to images A–C; (G, H) luminescence intensity across individual particles in images D and F. PBS containing 200 μM L012 and 1 mM hydrogen peroxide.

All samples were imaged in 10 mM

Scale bars are 20 μm.

The exposure time was

10 s. Figure 3. (A–E) Bright-field and ECL images of single rutile TiO2 particles in PBS solution and PBS solutions containing 0.1, 0.5, and 1 mM hydrogen peroxide; (F) correlation between the luminescence ratios of four nanoparticles and their hydrogen peroxide concentrations; (G) average luminescence ratio of three nanoparticles in the presence of 0.1 mM hydrogen peroxide during continuous recording for 90 min.

The

exposure time was 10 s; the scale bars are 20 μm. Figure 4. (A) Bright field; (B, C) ECL before and after stimulation of cells using PMA (B: before, C: after); (D) subtracted ECL images from image C and B after adjusting the contrast; (E) fluctuation of luminescence ratios of three nanoparticles after stimulation of the cell; (F) peak ECL intensity from 12 nanoparticles located under five individual cells.

The exposure time was 10 s; the scale bars are 20 μm.


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Figure 1.


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Figure 2.

A

C

B

6

5

1

3

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4 3

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H ECL Intensity / a.u.

200 100 0 1

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4

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ECL Intensity / a.u.

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


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G

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0.5 Conc./mM

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1.0

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Figure 4.


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BB

A

C

1 2 3

2.5 2.0 1.5 1.0 0

20

40 60 Time / s

80

D

C

3.0

E

Luminescence ratio

3.0 Luminescence ratio

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


F

2.5 2.0 1.5 1.0

1

2

3 Cell

4

5

TOC only


15


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16

Steady-State Electrochemiluminescence at Single Semiconductive

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