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Single Cell Electrochemiluminescence Imaging: From the Proof-of-Concept to Disposable Device-Based Analysis GIOVANNI VALENTI, Sabina Scarabino, Bertrand Goudeau, Andreas Lesch, Milica Jovic, Elena Villani, Milica Sentic, Stefania Rapino, Stephane Arbault, Francesco Paolucci, and Neso Sojic J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b09260 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Journal of the American Chemical Society

Single Cell Electrochemiluminescence Imaging: From the Proof-ofConcept to Disposable Device-Based Analysis Giovanni Valenti,1 Sabina Scarabino,2 Bertrand Goudeau,2 Andreas Lesch,3 Milica Jović,3 Elena Villani,1 Milica Sentic,2 Stefania Rapino,1 Stéphane Arbault,2 Francesco Paolucci,1,4 * Neso Sojic2*

1 2

Department of Chemistry ‘‘G. Ciamician’’, University of Bologna, Via Selmi 2, 40126 Bologna, Italy. University of Bordeaux, Bordeaux INP, ISM, UMR CNRS 5255, 33607 Pessac, France.

3

Laboratory of Physical and Analytical Electrochemistry, EPFL Valais Wallis, Rue de l’Industrie 17, CP 440, CH-1951 Sion, Switzerland

4

ICMATE-CNR Bologna Associate Unit, University of Bologna, via Selmi 2, 40126, Bologna, Italy.

KEYWORDS. Electrochemiluminescence, Single cell, Microscopy, Electrochemical imaging, Biosensors. ABSTRACT: We report here the development of coreactant-based electrogenerated chemiluminescence (ECL) as a surfaceconfined microscopy to image single cells and their membrane proteins. Labeling the entire cell membrane allows to demonstrate that, by contrast with fluorescence, ECL emission is only detected from fluorophores located in the immediate vicinity of the electrode surface (i.e. 1-2 µm). Then, to present the potential diagnostic applications of our approach, we selected carbon nanotubes (CNT)-based inkjet-printed disposable electrodes for the direct ECL imaging of a labeled plasma receptor over-expressed on tumor cells. The ECL fluorophore was linked to an antibody and enabled to localize the ECL generation on the cancer cell membrane in close proximity to the electrode surface. Such a result is intrinsically associated to the unique ECL mechanism and is rationalized by considering the limited lifetimes of the electrogenerated coreactant radicals. The electrochemical stimulus used for luminescence generation does not suffer from background signals, such as the typical auto-fluorescence of biological samples. The presented surface-confined ECL microscopy should find promising applications in ultrasensitive single cell imaging assays.

Introduction. Single cell microscopy has become a fundamental diagnostic tool for modern medicine and biology. The ultrasensitive quantitative detection of membrane proteins in single cells mostly relies on the imaging of fluorescent probes usually obtained by in situ hybridization and immunohistochemistry. The optimization of the imaging system to maximize the signal-tonoise ratio is still critical for numerous membrane proteins because they are present in very low quantities and intense illumination conditions are often necessary which may lead to an irreversible degradation of the biological samples. Despite the development of confocal, sheet illumination or single molecule microscopies which improve the signal-to-noise ratio and the imaging resolution, photoluminescence is intrinsically affected by the background signal of the sample matrix (e.g. auto-fluorescence), whose magnitude is often similar to the analyte specific signal. A high background signal can be efficiently minimized by chemiluminescence (CL) or bioluminescence (BL) methods.1,2,

Those techniques have been proposed successfully for ultrasensitive quantitative imaging on single cells and tissues using suitable substrates.3,4,5,6 Despite the high sensitivity of CLbased techniques the spatial resolution is strongly influenced by the analytical conditions. In particular, the product of the enzymatic reaction can diffuse in the solution before acting as a substrate for the CL reaction, resulting in a decrease of spatial resolution.7 In this context, the search for ultrasensitive and non-invasive techniques for functional imaging of single cells is still undergoing. Electrochemical techniques have been proved to provide high spatial resolution for single cell analyses. Imaging of single cells by scanning electrochemical microscopy (SECM) and by micro-metrically shaped arrays were reported to elucidate single cell motility,8 transformation,9 cell morphological transformation,10,11,12 oxygen consumption13 and metabolism.14,15 Herein we report the electrogenerated chemiluminescence (ECL) imaging of single cells. ECL is a powerful transduction technique gathering the advantages of the electrochemical sen-

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sitivity and the spatial resolution provided by fluorescence microscopy.16,17 In view of the electrochemical stimulus of the luminescence process ECL offers simplified instrumental setup, in fact the lightning source is not necessary. In addition remarkable characteristics were obtained for (bio)sensor applications, such as very low background and high sensitivity,18,19,20 good temporal and spatial resolution, robustness, versatility and low fabrication costs.16,17 All these characteristics make ECL, and in particular the coreactant approach,21 the most used transduction methodology in clinical assays dealing with very complex matrices, such as urine, blood or lysate,22,23,24 and leader companies developed and commercialized clinical analyzers for the immunoassays. Furthermore, the high versatility of the ECL transduction was largely applied to quantify circulating tumor cells25,26,27 leukemia cells28,29 or intracellular molecules,30 such as cholesterol31,32 or glucose,33 at the single cell level. An important breakthrough in the development of analytical ECL applications was its implementation as an imaging technique to visualize electrochemically single objects 34,35,36,37 and entities.38,39,40 In 2006, Amatore et al. combined the spatial visualization with ECL to observe with micrometric resolution concentration profiles of species generated over a double-band microelectrode assembly.41 Recently, the combination of ECL imaging and immunoassays resulted in an emerging approach, known as heterogeneous ECL, for visualizing micrometer-size objects onto an electrode surface.42 In this framework, our research groups recently reported the application of heterogeneous ECL for imaging microbeads.43,44,45 This strategy is based on the immobilization of an ECL active fluorophore (e.g., using an antibody specific reaction) onto the micro-object surface and on the indirect activation of the ECL emission by the electrogenerated radicals of an appropriate sacrificial coreactant, according to a well-known mechanism.18,46 In this configuration, both diffusion and stability of such radicals play a crucial role in determining, at each position of the microobject surface, the signal intensity and its time evolution.45 ECL imaging has, for instance, recently been proposed for the highly sensitive detection of protein/polypeptide residues in latent fingermarks 42 or for the quantification of cancer biomarkers.47,48,49 However, even if applied to different biological samples (as already mentioned, blood, urine, etc.), ECL has never been reported for the resolved imaging of single cells showing up the distribution of proteins on their membrane.

Scheme 1. Schematic principle for the ECL imaging of single cells

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In this context, we report the first, to the best of our knowledge, spatially-resolved ECL imaging of single cells (Scheme 1). Firstly, as a proof-of-concept, we chose a model cellular system, Chinese Hamster Ovary (CHO) cells, which was investigated on a glassy carbon electrode. Secondly, we extended our approach to show up the potential diagnostic applications of ECL cell imaging by selecting CNT-based inkjetprinted disposable electrodes for the direct mapping of a plasma membrane receptor over-expressed in tumor cells. Experimental Materials. Tri-n-propylamine (TPrA), phosphate buffered saline (PBS) solution (pH 7.4), streptavidin from Streptomyces avidinii, bis(2,2′-bipyridine)-4′-methyl-4-carboxybipyridineruthenium N-succinimidyl ester-bis(hexafluorophosphate) [Ru(bpy)2(mcbpy-O-Su-ester)(PF6)2], paraformaldehyde (PFA) and tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]Cl2*6H2O) were from Sigma-Aldrich and used without any further purification. Iscove's modified Dulbecco's medium, fetal bovine serum, PBS 1X, trypsin and penicillin/streptomycin 100 U/mL were from Gibco. Biotin-X was from Fisher Scientific. DMSO was from Invitrogen. Phosphate buffer (PB) 0.2 M (pH 6.8) was obtained by mixing 0.2 M of sodium phosphate monobasic dihydrate (NaH2PO4*2H2O) and 0.2 M of sodium phosphate dibasic (Na2HPO4), both from Sigma-Aldrich and used as received. Cetuximab antibody was obtained from the Erbitux. Ruthenium-butanic acid-ester (Ru(bpy)2-bpy-CO-OSu) was purchased from Roche Diagnostics GmbH (Mannheim, Germany) and used as received. Amicon centrifugal filters were obtained from Millipore (Vimodrone, Italy) and dialysis membranes (12000-14000 Da cut-off, in regenerated cellulose) were obtained from Spectrum Lab (Rancho Dominguez, CA, USA). ITO was purchased from Kuramoto Seisakusho Co. Ltd. (Tokyo, Japan). Carbon nanotube ink CNTRENE (Brewer Science), Ag ink Silverjet DGP-40LT-15C (w/w 35%, Sigma-Aldrich) and dielectric ink EMD6201 (Sun Chemical) were used for inkjet printing. Polyethylene terephthalate sheets (PET, 125 µm thick) were obtained from Goodfellow as substrates for inkjet printing. Inkjet printing of CNT electrodes. CNT electrodes were fabricated by using an X-Serie CeraPrinter (Ceradrop) equipped with three parallel printheads, i.e., two Q-Class Sapphire printheads with 256 nozzles (QS-256; Dimatix Fujifilm) and one disposable DMC-11610 cartridge (Dimatix Fujifilm). An integrated UV LED FireEdge FE300 (380-420 nm; Phoseon Technology) allows simultaneous printing and UV photo-polymerization of the dielectric ink using the Dimatix cartridge. The post-processing station of the printer contains a PulseForge 1300 photonic curing system (Novacentrix) for the rapid in-line thermal curing of the Ag and CNT patterns. All printing parameters, such as jetting frequency, pulse forms for the piezoelectric actuation and number of active nozzles were optimized for high resolution printing. Firstly, Ag patterns for the electrical connections and as conductive traces were printed and cured. Secondly, a CNT pattern was printed that only slightly overlapped with the Ag pattern for the electrical connection in order to form a standalone CNT electrode on PET. After photonic curing of the CNTs, the UV curable dielectric ink was printed as insulator to define the active CNT working electrode area and to cover the Ag trace. The CNT electrodes could directly be used.


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Labeling of streptavidin with ruthenium complex (SA@Ru). A solution of 100 µL of ruthenium complex (10 mg/mL) in anhydrous DMSO, 100 µL of streptavidin (1 mg/mL in PBS) and 800 µL PBS was prepared. The solution was vortexed for 5 h at 4 °C and dialyzed overnight under stirring at 4 °C. Labeling of antibody (Ab) with ruthenium complex (Ab@Ru). At 1 mg/mL of antibody (Ab, Cetuximab) in PBS solution, pH 7.8, 85 molar equivalents of EDC, NHS and Ru(bpy)2-bpy-CO-OSu were added. In this case we prefer to work without DMSO that might partly denature or modify the antibody. The solution was gently shaken for 90 minutes at room temperature and then exchanged to pH 7.4 by dialysis using a 12000-14000 Da cut-off membrane and maintained overnight at 4°C. In order to remove the unreacted dye complex, the solution was centrifuged with Millipore Amicon® Ultra 0.5 µm centrifugal filter devices, with 50000 cut-off membrane and refilled with fresh PBS.50 Cells culture. CHO-K1 cells were from Public Health England (HPA) Culture Collections and supplied by Sigma (85051005). Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 10% Fetal Bovine Serum and Penicillin/Streptomycin 100U/mL were used to grow cells in an incubator. CHO-K1 cells were trypsinized, plated on glassy carbon electrodes and incubated 48 h with culture medium at 37°C, 5% CO2. MCF10A cells (ATCC: crl-10317) were cultured in (1:1) Dulbecco’s Modified Eagle’s Medium (DMEM)/Nutrient Mixture F-12Ham (Gibco-Life Technologies Corporation) supplemented with 5% horse serum, L-Glutamine 1mM, Penicillin/Streptomycin 50 U/mL, 20 ng/mL epidermal growth factor (EGF), 50 ng/mL cholera toxin, 500 ng/mL hydrocortisone and 0.01 mg/mL insulin (Gibco-Life Technologies Corporation). Cells were passed upon trypsin digestion and cultured at 37°C, 5% CO2. 30000 MCF10A cells in 2.5 mL of complete medium were plated in a 3.5 cm Petri dish containing CNT electrode and incubated over night at 37°C, 5% CO2 before the recognition step. Biotin streptavidin and antibody recognition on adherent cells. Before ECL experiment, CHO-K1 cells were fixed 10 min with PFA 4%, labeled with biotin X 11 µM and streptavidin-ruthenium complex solution SA@Ru (0.1 mg/mL). The optimized procedure for the antibody recognition on MCF10A consisted in the following series of operations: (i) cells were cultured in adhesion to the electrodes as described in the cell culture section; (ii) before use, the medium was removed and the cells were washed two times with PBS solution; (iii) successively, the cells were incubated for 7 min at room temperature with a solution containing Ab@Ru 0.15 nM in PBS. The unreacted Ab@Ru was removed by washing two times with PBS. (iv) The cells/Ab@Ru were fixed using PFA 0.5% for 20 min. In both cases cells were rinsed thoroughly with PBS 1X between the different steps. ECL detection. The ECL measurements were carried out in a phosphate buffered solution (PBS 0.2 M, pH 6.8) using TPrA as an oxidative-reduction sacrificial coreactant. The counter electrode was a Platinum spiral and the reference electrode was a homemade Ag/AgCl/KCl 3M. The ECL signal was measured with a photomultiplier tube (PMT, Hamamatsu

R4220p) placed above the electrochemical cell at constant distance and inside a dark box. The voltage supplied to the PMT was in the range 550-750 V. The light/current/voltage curves were recorded by collecting the preamplified PMT output signal (by an ultralow-noise Acton research model 181) with the second input channel of the ADC module of the AUTOLAB instrument. Chronoamperometry technique was used for ECL detection: E1=0 V, t1=2 sec ; E2=1.35 V, t2=6 sec. Imaging instrumentation. The ECL/optical/PL imaging was performed in a PTFE (Teflon) homemade electrochemical cell, described in Figure S1. The ECL imaging cell was used in a three-electrode configuration, in which the CNT electrode acted as working electrode, a Ag wire as quasi-reference electrode and a Pt wire as counter electrode. For microscopic imaging, an epifluorescence microscope from Nikon (Chiyoda, Tokyo, Japan) equipped with ultrasensitive ElectronMultiplying CCD camera (EM-CCD 9100-13 from Hamamatsu, Hamamatsu Japan) was used with a resolution of 512 × 512 pixels with a size of 16 µm × 16 µm. The microscope was enclosed in a homemade dark box to avoid interferences from external light. It was equipped with a motorized microscope stage (Corvus, Märzhauser, Wetzlar, Germany) for sample positioning and with long distance objectives from Nikon (10x/0.30 DL17, 5mm, 20x/0.40 DL13mm). The integrated system also included a potentiostat from AUTOLAB (PGSTAT 30) suitable to provide the needed potential for the ECL triggered reaction.

Results and discussion. The initial goals of this work were to demonstrate the imaging of a single cell by ECL and study its occurrence at the cell membrane. For this, we labeled the cell membrane with a model luminophore Ru(bpy)

(bpy = 2,2’-bipyridine) via a biotin-streptavidin link. TPrA was added to the medium and used as an oxidative-reductive coreactant. The mechanism of ECL generation with the above tandem system has been deciphered18,41 and is at the basis of major commercial applications of ECL. The electrode material51 is also crucial for an efficient ECL generation. Carbon-based materials lead very efficiently to strong and reproducible ECL signals, thanks to the relatively faster kinetics for the amine oxidation and lower susceptibility to fouling than noble metal electrodes.51 To demonstrate the proof-of-concept of our ECL imaging approach, we selected first glassy carbon (GC) electrodes. CHO cells were then grown onto the GC electrode and incubated with biotin X which reacts with primary amino groups of proteins under mild conditions.52 Then the biotin groups were reacted with streptavidin-modified Ru(bpy)

-moieties (SA@Ru) which thus remained attached to cell membrane proteins all along ECL measurements (Scheme 1a). The biotinylation procedure is a simple and efficient way to label specifically cell surface proteins, whereas endogenous proteins are not labeled.52 Figure 1 compares the PL and ECL images of labeled CHO cells recorded over the same region of interest of the GC electrode. The PL image highlights the spatial distribution of the SA@Ru labels thus visualizing the entire cell and its different structures. Typical cells were about 30-70 µm in diameter, similarly to when observed in usual culture conditions (i.e., in Petri dish). The cellular membrane which defines the cyto-



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plasm limits was clearly visible as well as the pseudopods ensuring the adhesion on the GC surface (Figure 1a). The nucleus, localized generally in the middle of the cell, appeared slightly darker. Figure 1b reveals the ECL light emitted by the SA@Ru labeled cells when an anodic potential of 1.35 V was applied to the GC electrode in a TPrA solution. Notice that, by contrast with PL, ECL was not emitted by the entire cell surfaces but was essentially localized at the cell borders. Such a

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result, intrinsically associated to the unique mechanism that generates ECL in the present system, deserves further analysis and will be discussed below. Comparison with PL (Figure 1c) also shows that ECL allows visualizing a significantly larger spatial extension of the cell contour. This is likely a result of the better signal-to-background ratio which characterizes ECL, in which auto-fluorescence phenomena are avoided.

Figure 1. PL (a) and ECL (b) images of CHO cells grown on a GC electrode. SA@Ru labels were attached to the biotinylated proteins of the cellular membrane. (c) Overlay of the PL (green) and ECL (red) intensity on the same region of interest. ECL was recorded in phosphate buffer solution (pH = 7.4) containing 100 mM TPrA by applying 1.35 V. Scale bar: 20 µm.

This first series of results demonstrated the validity of the methodological approach. However, GC material can hardly serve for the fabrication of practical disposable analytical devices for ECL imaging of cells or tissues surfaces. Optimal electrode materials might in fact combine good electrical conductivity with optical transparency, which would allow the complementary easy visualization of cells by traditional optical microscopy or, alternatively, enable the integration of the photosensing element into the same electrodic platform. We have recently shown that, when compared to traditional transparent electrodes based on metal oxides (e.g., ITO), carbonbased nanomaterials and in particular carbon nanotubes (CNTs) may be ideal platforms for the present application.51,43 Indeed, they display high stability and enhanced electrocatalytic properties versus ITO for the generation of the coreactant-based radicals involved in the ECL generation. In such a context, CNT-based transparent electrodes that can be reproducibly fabricated at reasonable costs would therefore represent the best possible choice. In this direction, inkjet printing has recently emerged as a technology approaching closely the industrial production level for thin layers of functional materials.53,54,55 This technique is a digital, mask-less and contactless material deposition technique, where picoliter droplets are ejected from up to several hundred parallel nozzles. Other transparent carbonaceous materials such as carbon nanoribbons and nanohorns could potentially be used as far as material deposition techniques allow the fabrication of homogeneous transparent and conductive layers. For the present application, CNT working electrodes made of short double-walled CNTs were deposited on a transparent polyethylene terephthalate (PET) substrate (Figure 2a and Figure S2a,b). The CNT elec-

trodes showed a homogeneous surface coverage, high light transmission (>90% @500-700 nm, Figure S2a) with fast electron transfer kinetics (Figure S2c). 56,50 ,43 The efficiency of their electrochemical response was proved by cyclic voltammetry (Figure S2c) as well as by their higher ECL activity compared with standard ITO (Figure S2d and Figure S3).57,33,31,32,43 The biocompatibility of the patterned CNT substrates was also evaluated: MCF10A cells were cultured on the inkjet-printed CNT electrodes and cell viability checked after 24h, 48h and 72h from the plating time, showing that the cells adhering on the CNTs were viable. Cell growth curve on the inkjet-printed CNT substrates was found to be comparable with standard Petri dish cultures (Figure 2c). The comparable cell adhesion onto the printed dielectric ink and the PET substrate is therefore highly encouraging for the use of such substrates in disposable devices for cell visualization by ECL (Figure 2b). To further explore the potential of the above strategy, we tested the application of the CNT substrates for the ECL detection and visualization of a cancer marker on adherent cells. We targeted the epidermal growth factor receptor (EGFR), a plasma membrane receptor over-expressed in several cancer cells (and in MCF10A cells in particular).58 A monoclonal antibody, labeled with Ru(bpy)

(Ab@Ru), was used for the efficient and selective targeting of the EGFR (Scheme 1b).59,60 The cells were plated on the CNTs substrates and incubated over night; after the labeling with the EGF antibody, the cells were fixed in PFA.


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The effective bio-recognition of the Ab@Ru was tested by analyzing the photoemission behavior from a single MCF10A cell while the CNT electrode was unbiased. Figure 2d shows a typical PL image displaying an intense and homogeneous signal from the entire cell demonstrating the immune recognition of the Ab@Ru with the EGFR on the cell membrane. The excitation light source was then switched off and a positive potential of 1.35 V was applied, in the presence of 200 mM TPrA, to initiate the ECL process while carrying out the simultaneous image acquisition. Figure 2e shows the ECL mapping of EGFR on the same cell. Moreover, the integrity of analyzed cells was confirmed, after the ECL experiments, by the nucleus labeling with DAPI (4',6-diamidino-2phenylindole) in PL (Figure S4).

Figure 2. The transparent CNT-based disposable electrode (a) and optical image of the electrode border with the MCF10A cells (b). Cells vitality (c) on the inkjet-printed CNT electrodes (red) and on the standard Petri dish cultures (blue) after 24, 48 and 72 h. PL (d) and ECL (e) images of a MCF10A cell labeled with Ab@Ru in phosphate buffer 0.2 M with 200 mM TPrA and adhered on an inkjet-printed CNT electrode. Potential applied 1.35 V. All potentials are reported vs. Ag/AgCl/KCl 3M.

Similarly, to results obtained with the CHO cells, the emission profiles of the ECL images recorded in the present case differ significantly from the PL ones (see the comparison on Figure 3). As in the former case, emission is clearly lower in the central region of the cell. A physical justification of such a different spatial distribution of light emission has to be found in the intrinsic mechanism of ECL generation, vis-à-vis PL, that applies to cases where diffusion and direct oxidation of the luminophore at the electrode surface are impeded, as in the present configuration. The prevailing mechanism for ECL generation would thus involve exclusively the radicals obtained by the anodic oxidation of TPrA, thought the following mechanism (heterogeneous ECL), originally proposed to explain excitation of Ru-complexes covalently attached onto microbeads for immunoassays:46,61,45 TPrAH+ TPrA + H+

(1)

TPrA - e TPrA●+

(2)

TPrA●+ TPrA● + H+

(3)

TPrA● + Ru(bpy)

P1 + Ru(bpy)

(4)

TPrA●+ + Ru(bpy) TPrA + Ru(bpy)

*

(5)

Ru(bpy)

∗ Ru(bpy) + hν

(6)

where P1 is the product of the homogeneous TPrA● oxidation. Briefly, the starting step is TPrA oxidation to TPrA•+ which undergoes further deprotonation to TPrA•, a strong reductant

(reaction 3). This radical reduces Ru(bpy)

to Ru(bpy) (reaction 4) while pristine TPrA●+ oxidizes Ru(bpy) to generate the excited state Ru(bpy) ∗ (reaction 5), which relaxes to the ground state generating the ECL signal.

Figure 3. PL (a and c) and ECL (b and d) profiles of a CHO cells labeled with SA@Ru (a and b) and of a MCF10A cell labeled with Ab@Ru (c and d) in phosphate buffer 0.2 M with 200 mM TPrA. Potential applied 1.35 V. All potentials are reported vs. Ag/AgCl/KCl 3M.

Heterogeneous ECL is intrinsically a surface-confined phenomenon since it is limited by the access of the electrogenerated radicals to the luminophore site. This is not likely the case for the internal region of the cells where the luminophore may be located at distance also exceeding some micrometers from the electrode. Emissions would then be mostly localized in the proximity of the cell border, i.e., close to the electrode surface. The accessibility issue can be resolved by permeabilizing the cells as it is classically performed, for example, in immunofluorescence or Western blot.62 AFM experiments aimed to study the cell profiles (Figure S5) evidenced in fact, for the CHO cells, nuclei regions as high as 1-2 µm and significantly thinner membranes in the range of 800 nm. These topographic results are in agreement with similar AFM studies on the same cell line.63 The above hypothesis was then quantitatively assessed by numerical digital simulation that was used to model the spatial extension of the ECL-emitting layer over a cell, considered herein as an insulating object.45 According to the reaction scheme (1)-(6), the generation of the excited state requires that both the strong reducing radical TPrA• and the strong oxidizing radical cation TPrA•+, generated at the electrode, coexist in proximity of the membrane-bound luminophore. The efficiency of such a mechanism depends strongly on the lifetime of



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the radical species. The exquisite complexity of the living cell (i.e., shape, topography, adhesion, composition, activity, etc.) makes it a remarkably multifaceted and variable object that may affect ECL at each step of the process, from the initial

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electrochemical stimulation to the final photochemical relaxation, including the intermediate homogeneous chemical reactions.

Figure 4. Cross-section view of the simulated ECL profile at an MCF10A cell in 0.2 M PBS with 200 mM TPrA. a) TPrA radical cation, b) TPrA radical concentration profiles and c) the ECL intensity. The inset shows the integrated ECL intensity along the cell profile. All the details on the simulation parameters are fully described in the SI.

Given the complexity of the mechanism for the ECL generation, finite element method analysis was adopted.64,65 In the simulations, we have taken into account the above mechanistic scheme for TPrA oxidation at the electrode surface, to estimate the diffusion and reactivity of the resulting coreactant radicals TPrA•+ and TPrA• and their reaction with the immobi66,61,67 lized Ru(bpy)

In the simula labels (reactions 1-6). tions, TPrA diffusion through the cell membrane was not allowed (thus excluding its oxidation in the electrode region covered by the cell) and the Ru(bpy)

moieties were as sumed to be evenly distributed over the cell. In Figure 4 the results of steady-state simulation (see SI for the details, Tables S1-S5 and Figures S6-S8) are reported. ECL generation is governed by the concentration profiles of TPrA radical species at the surface of the labeled cell (see Figure 4a and 4b). In particular, we analyzed the ECL intensity over the lateral section of the cell where the luminophores are positioned at ever increasing distance from the accessible working electrode surface (at x < 0). From the ECL simulation profile, we can observe that the spatial location and extension of the ECL-emitting region is in fact strictly confined to a narrow region in proximity of the cell/electrode border. The ECL intensity increases progressively and reaches a maximum at a lateral distance x ∼ 1.5 µm and decreases virtually to zero at

longer distances (x > 4 µm). Simulations would therefore substantiate the hypothesis that diffusion, along with a finite lifetime of TPrA•+, are limiting the signal generation over the inner region of the cell membrane. In other words, only a limited fraction of the sample membrane which is immediately adjacent to the electrode contributes to the ECL signal. Conceptually, this is reminiscent of Total Internal Reflection Fluorescence (TIRF) microscopy where the analytical signal is associated to the evanescent wave which decays exponentially into the sample and only fluorophores that reside within ∼200 nm from the surface may contribute to the fluorescence emission. Similarly, in ECL microscopy of cells, emission from molecules located at more than 1-2 µm from the electrode surface is almost completely absent. Finally, as already mentioned, it is important to notice that the ECL imaging of both cell lines was performed after the fixation with PFA due to the high concentrations of TPrA used. However, the use of new, recently reported, less toxic coreactants such as diethanolamine68 or common biological buffers containing aliphatic tertiary amines69 might open up this application for the analysis of living cells in culture. This approach is currently under investigation in our laboratories.


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Journal of the American Chemical Society (2)

Conclusions

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In conclusion, we have demonstrated the potentiality of ECL as an alternative surface-confined microscopy to image single cells and their membrane proteins. Firstly, the entire cell membrane was labeled to study the distribution of the ECL intensity. This has demonstrated that ECL emission is confined to the region of the cell membrane in the immediate vicinity of the electrode surface. Disposable inkjet-printed CNT electrodes provided a good biocompatibility for the cells and also superior transduction intensity compared to the current standard materials such as ITO. To observe ECL on cell membranes, biotin-streptavidin and immunochemistry strategies could be both used for labeling, allowing for instance to image the important membrane receptor EGFR on cancer cells. The ECL generation on two cell types tested herein occurred next to the uncovered electrode surface. The electrochemical stimulus used for luminescence generation does not suffer from background signals, such as the typical auto-fluorescence in biological samples. The reported results contribute to a better understanding of the mechanisms and operating conditions for cell analysis based on ECL and pave the way for the development of new surface-confined microscopy and of ultrasensitive single cell imaging assays.

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ASSOCIATED CONTENT

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Supporting Information

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AFM image of single cell, ECL properties of disposable inkjetprinted electrodes and simulation parameter details [Figures. S1S8 and Tables S1-S5]. The Supporting Information is available free of charge on the ACS Publications website.

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AUTHOR INFORMATION

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Corresponding Author * [email protected] (F. P.); [email protected] (N.S.)

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ORCID Giovanni Valenti: 0000-0002-6223-2072 Andreas Lesch: 0000-0002-4995-2251 Stefania Rapino: 0000-0001-6913-0119 Stéphane Arbault: 0000-0002-4994-2213 Francesco Paolucci: 0000-0003-4614-8740 Neso Sojic: 0000-0001-5144-1015

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ACKNOWLEDGMENT

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We thank the University of Bologna, Italian Ministero dell’Istruzione, Università e Ricerca (FIRB RBAP11C58Y, PRIN-2010N3T9M4), FARB, Fondazione Cassa di Risparmio in Bologna and the Agence Nationale de la Recherche (NEOCASTIP ANR-15-CE09-0015-03). We thank Dr Riccardo Marega (University of Namur) and Prof Davide Bonifazi (Cardiff University) for the useful discussion on the Cetuximab antibody.

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