High-Density Droplet Microarray of Individually Addressable

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High-Density Droplet Microarray of Individually Addressable Electrochemical Cells. Huijie Zhang, Tobias Oellers, Wenqian Feng, Tarik Abdulazim, En Ning Saw, Alfred Ludwig, Pavel A. Levkin, and Nicolas Plumeré Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 09 May 2017 Downloaded from http://pubs.acs.org on May 12, 2017

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Analytical Chemistry

High-Density Droplet Microarray of Individually Addressable Electrochemical Cells. Huijie Zhang,1 Tobias Oellers,2 Wenqian Feng,3 Tarik Abdulazim,1 En Ning Saw,1 Alfred Ludwig,2 Pavel A. Levkin,3,4* Nicolas Plumeré1* 1

Center for Electrochemical Sciences - CES, Ruhr-Universität Bochum, Universitätsstr. 150, 44780, Bochum, Germany Chair of MEMS Materials, Institute for Materials, Faculty of Mechanical Engineering, Ruhr-Universität Bochum, Universitätsstr. 150, 44780, Bochum, Germany 3 Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany 4 Institute of Organic Chemistry, Karlsruhe Institute of Technology (KIT), 76021 Karlsruhe, Germany 2

ABSTRACT: Microarray technology has shown great potential for various types of high-throughput screening applications. The main readout methods of most microarray platforms, however, are based on optical techniques, limiting the scope of potential applications of such powerful screening technology. Electrochemical methods possess numerous complementary advantages over optical detection methods, including its label-free nature, capability of quantitative monitoring of various reporter molecules, and the ability not only to detect but also to address compositions of individual compartments. However, application of electrochemical methods for the purpose of high-throughput screening remains very limited. In this work, we develop a high-density individually addressable electrochemical droplet microarray (eDMA). The eDMA allows for the detection of redox-active reporter molecules irrespective of their electrochemical reversibility in individual nanoliter-sized droplets. Orthogonal band microelectrodes are arranged to form at their intersections, an array of three-electrode systems for precise control of the applied potential and enable direct read-out of the current related to analyte detection. The band microelectrode array is covered with a layer of permeable porous polymethacrylate functionalized with a highly hydrophobic-hydrophilic pattern forming spatially separated nanoliter-sized droplets on top of each electrochemical cell. Electrochemical characterization of single droplets demonstrates that the underlying electrode system is accessible to redox-active molecules through the hydrophilic polymeric pattern and that the non-wettable hydrophobic boundaries can spatially separate neighboring cells effectively. The eDMA technology opens the possibility to combine the high-throughput biochemical or living cells screenings using the droplet microarray platform with the sequential electrochemical read-out of individual droplets.

Living cell microarrays for systematic screening of large chemical and genomic libraries are important in many fields, including drug discovery, toxicology and basic cell biology.1–4 The classical read-out to visualize cell responses relies on optical methods, which typically need labeling with fluorophores. In parallel, electrochemical detection principles based on microelectrodes have emerged as label-free and sensitive tools for direct and continuous monitoring of products generated or molecules consumed by living cells, while maintaining the biological and environment conditions unaffected.5–15 While such electrochemical methods were until now predominantly applied to study a single living cell or a single population of cells, their intrinsic advantages also motivated the integration of electrodes as sensing components into microarrays for the purpose of high-throughput screening of cell libraries. To this end, each testing point in the microarray needs to be associated to individually addressable electrodes. In this case, the wiring of the electrode to an external circuit defines the maximum density of the microarray.16 The simplest and most robust design for high density wiring is possibly the crossbar assembly developed for nanoelectronic circuitry.17,18 A similar architecture based on arrays of band electrodes was proposed

by Ino and Matsue for the read-out of living cell microarrays.19 In this system, two parallel groups (rows and columns) of band microelectrodes are positioned orthogonally to each other. The crossing points of two band electrodes can be addressed individually by contacting the respective row and column electrodes. A remarkably high density of testing points can be achieved since multiple measurement points share the same wires to the external circuit. Several addressable microarray electrode systems have been reported since then, including orthogonal microband electrode arrays with microwell,20 vertically separated electrode array,21 interdigitated electrode array,22,23 ring–ring electrode array,24,25 ring-disk electrode array,26 nanocavity crossbar arrays27 and droplet array on comb-type interdigitated ring array electrodes.28 In these systems, the electrochemical signal is based on the redox cycling of chemically reversible redox couples between the two electrodes which results in signal amplification related to multiple oxidation and reduction processes for each analyte molecule. However, with this measurement principle, the detection of irreversible redox species, which include numerous important analytes such as O2,29 neurotransmitters,30 reactive oxygen and nitrogen species,6 can only be achieved with particular set-ups as illustrated in the work by Ino et al propos-

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ing diffusive molecular competition for electrochemical detection of O2 to evaluate the respiratory activity of cells.31 Recently, Kelley et al. extended the concept of the intersecting band electrodes to a three electrode system based on a pair of parallel counter and reference electrodes associated to an orthogonal working electrode. In this case, the measurement points were connected by microchannel solution-based circuits for sample loading. 32 While the latter system was developed as a DNA sensor, it is in principle suitable for detection of any electroactive species, irrespective of their electrochemical reversibility. However, its conceptual design based on numerous testing points sharing the same liquid channel, makes it less suitable for microarray applications that are sensitive to cross-contamination. In other words, a simple and general electrochemical read-out method specifically designed for microarray screening remains to be developed. Here we report the first microarray platform that combines the advantages of electrochemical read-out based on threeelectrode systems and of immunity to cross-contamination based on compartmentalization into separated microdroplets formed on a highly hydrophobic-hydrophilic pattern. The electrochemical microdroplet array (eDMA platform) include two sets of parallel Au bands used as pseudo-reference and counter electrodes, and another set of Au bands arranged orthogonally serving as the working electrodes. This three sets of band microelectrodes (Figure 1A) are covered by a layer of chemically functionalized permeable porous polymethacrylate to form an array of hydrophilic wettable squares of 600 µm separated by 150 µm wide hydrophobic, water-repellent barriers.33–37 The extreme difference in hydrophilicity between the spots and the background leads to the effect of discontinuous dewetting,37 which enables a single-step formation of hundreds of droplets in every hydrophilic spot by simple dragging of a “source droplet” over the array. The non-wettable hydrophobic boundaries in the polymeric pattern serve a dual function in blocking both ion transport in the porous polymer and formation of separated droplets on the surface (Figure 1B). Therefore, the band electrodes intersecting in each microdroplet represent a three-electrode electrochemical cell (Figure 1C) that can be individually addressed for electrochemical read-out irrespective of the electrochemical reversibility of the redox-active molecules. Due to the separation of the microdroplets, this set-up is intrinsically immune to crosscontamination.

MATERIALS AND METHODS Fabrication of the eDMA. The fabrication of the Au band structures and the polymer pattern deposition are described in detail in the supporting information. Electrochemical measurements with the eDMA. Electrochemical experiments were carried out by using a Reference 600 potentiostat (Gamry Instruments, Warminster, PA). For the characterization of the whole array, the band microelectrodes were connected by means of conductive copper tape to the potentiostat. The reference electrodes was either the internal Au bands or an external Ag/AgCl (KCl 3M) electrode. The counter electrode was either an internal Au band or an external Pt wire. To address individual droplets on the array, the three corresponding Au band electrodes were used as working, counter and pseudo-reference electrodes. All single droplet experiments were performed in water saturated atmosphere.

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Figure 1. Schematic representation of the electrochemical droplet microarray. (A) Overview showing the contact pads for the working electrodes (WE) and counter electrodes (CE) on the left and shared contact pad for the pseudo-reference electrode (pRE) on the right. (B) Excerpt of the droplet array after wetting of the porous polymer layer. (C) Cross-section of a single electrochemical droplet cell covered with a layer of porous polymer with defined water-impermeable hydrophobic boundaries (white) surrounding water-wettable hydrophilic porous regions. The electrochemical cell including the intersection of the Au band microelectrodes (yellow) separated by an insulator layer (blue band) is embedded into the wetted porous polymer matrix (transparent blue).

RESULTS AND DISCUSSION Fabrication of the eDMA. Three sets of Au band electrodes serving as counter, working and pseudo-reference electrodes are made by UV photolithography (Figure 2A and Figure S1) and physical vapor deposition in a lift-off process. The silicon substrate is first coated with two sets of parallel band electrodes (80 µm and 200 µm in width) with the narrow Au band serving as pseudo-reference electrode (pseudo-RE). A third set of orthogonally arranged band electrodes (72 µm in width) is deposited in a subsequent photolithographic step and separated from the two sets of bottom electrodes by an insulating layer made of SU-8 photoresist. The intersections of the 20 top electrodes with the 20 sets of bottom electrodes yield up to 400 possible electrochemical cells. An alkyne-functionalized porous poly(2-hydroxyethyl methacrylate)-co-(ethylene dimethacrylate) (HEMA-EDMA) polymeric film is formed on top of the electrode array.38 After photopatterning via sequential thiol-yne click reactions, the highly hydrophobic boundaries as well as highly hydrophilic spots (600  600 µm squares) are produced on the polymer film (Figure 2B). The scanning electron microscopy (SEM) images of the crosssection of the array reveals the various layers on the Si / SiO2 substrate. The layer of polymer film is homogenous in its film thickness (35 µm) and displays a porous structure without cracks or pinholes (Figure 2C). The polymer is tightly connected to the underlying Au band electrode (100 nm thick) which is deposited on the 11 µm thick SU-8 insulator (Figure 2D).


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Figure 2. Fabrication of the electrochemical droplet microarray. (A) Step-wise deposition of the orthogonally arranged Au band electrodes separated by an SU-8 insulator layer. (B) Deposition of the porous polymer layer and patterning of highly hydrophilic spots separated by highly hydrophobic barriers for droplet confinement on top of each band electrode intersections. (C) SEM image of the cross-section of the band electrode array covered with the porous polymer layer and (D) close-up of the interfaces between the SU-8 insulator, the Au band electrode and the porous polymer film.

Individual droplets (about 20 nL each35) spontaneously form on the eDMA by exposing the substrate to an aqueous solution (Figure 3). Droplet deposition can be performed by various methods such as i) by dipping the complete substrate into an aqueous solution (Figure 3B), ii) by rolling a larger droplet of solution across the surface (Figure 3C and S2) or by dropspotting (Figure 3D). Due to the extreme difference in wettability between the highly hydrophilic spots and the highly hydrophobic barriers (see water contact angle, Figure 3E), the aqueous microdroplets remain exclusively located in the hydrophilic spots making them also more transparent due to the decreased light scattering (Figure 3B-D). Direct indication for the droplet separation is given by the optical images of the cross-section of the eDMA polymer film (Figure 3F). While the dry polymer appears as a colorless opaque film, the waterwetted films reveal the position of the droplets. To better visualize the droplets and possible cross talks, we added a red dye (neutral red) to the water solution. After droplet drying, the red color is located solely in the hydrophilic regions while the hydrophobic barriers are not accessible to the aqueous solutions and therefore remain colorless.

Figure 3. Droplet formation. Optical photograph of (A) the dry polymer film coated electrode array and of (B) the array of the individual droplet cells (600 µm × 600 µm, ~20 nL each) on the polymer coated electrode array obtained by dipping the complete substrate in aqueous solution followed by shake-off of the excess liquid. (C) Droplet formation by rolling a large droplet of aqueous solution across the surface. (D) Close-up of a droplet formed by single drop-spotting. The hydrophilic region appears transparent upon wetting. (E) Water droplet on a uniformly modified superhydrophobic polymer film. (F) Cross-section view of the patterned polymer coated electrode array in the dry state (top), covered with pure water droplet (middle) and after drying of water droplets containing neutral red (bottom).

The volume of the droplets depends on the size of the hydrophilic spots and can be varied between 1 nL for 200 µm spots and about 100 nL for 3 mm spots.36 The droplet geometry and their nanoliter-sized dimension were previously demonstrated to be suitable for culturing living cells and performing cell screening experiments.35–37 Electrochemical behavior of the eDMA. The individual microdroplets on top of the intersection of the orthogonal Au bands form 3-electrode electrochemical cells of microscale dimensions. The electrochemical behavior of the uncoated band electrodes and of the eDMA is first investigated in aqueous electrolytes (0.5 M H2SO4 and 1 M KNO3). Cyclic voltammograms of the uncoated band electrodes display a current response attributed to Au-oxide formation and reduction which is characteristic of Au surfaces (Figure S3A-B). After modification of the Au electrode array with the polymer film, the electrochemical behavior is mostly unchanged in comparison to the one of the bare electrodes (Figure S3C-D). This demonstrates that the hydrophilic regions of the porous polymer are fully solvated by the aqueous electrolyte. CVs from a single droplet containing aqueous KNO3 show a wide electrochemical window of about 1.5 V (Figure S4). Faradaic cur-



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rents at potentials below -0.5 V vs the Au pseudo RE may be attributed to O2 reduction. In order to test the diffusive mass transport of electroactive molecules through the polymeric matrix and charge transfer with the underlying Au band electrodes, we select three redox probes as model systems: i) the negatively charged iron hexacyanoferrate, ii) the positively charged ruthenium hexamine and iii) the neutral ferrocene dimethanol. The cyclic voltammograms of each redox probe were recorded in single droplets formed using the rolling droplet method. All three redox systems display currents corresponding to their respective oxidation and reduction (Figure 4A-C), thus demonstrating the permeability of the polymer film irrespective of the charge of these model systems. Moreover, the CV of ferrocene dimethanol (Fc(MeOH)2) remains mostly unchanged after performing an electrochemical cleaning process (Figure S5A). Therefore, the electrodes as prepared by lithography and modified with the porous polymer layer are directly suitable for electrochemical applications.

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cient or the concentration of the redox probe. The effective diffusion coefficient (Deff) in the polymer film may be lowered due to the void fraction (ε) and tortuosity (τ) as Deff = ε Dfree / τ which defines the diffusion in porous structures 39,40 and typically imposes a value for Deff lower than the diffusion coefficient in bulk solution (Dfree). The concentration of the redox probe within the film may be lower than in the bulk of the electrolyte due to partition of the redox-active molecule. Nevertheless, we can conclude that the hydrophilic polymer film does not significantly block the diffusion of the redox active molecules.

Figure 4. Electrochemical characterization of single droplets containing redox-active species. Cyclic voltammograms of (A) Fc(MeOH)2; (B) K3Fe(CN)6; (C) Ru(NH3)6Cl3. The concentration of all redox-active species is 1 mM in KNO3 (1 M). The narrow top Au band was used as WE. The scan rate is 100 mV.s-1.

However, the current response for all three redox probes at the scan rate of 100 mV.s-1 (Figure 4) deviates from the behavior expected for a simple reversible electron transfer and semiinfinite planar diffusion. While a coupled follow-up homogeneous reaction involving O2 reduction may contribute to an enhanced cathodic current in the case of Ru(NH3)6Cl3, the main reason for the shape of the CVs in Figure 4 may be related to the microelectrode dimensions. Direct evidence for contributions from hemispherical diffusion as well as quantitative information on the diffusive mass transport properties within the polymer film is gained from recording cyclic voltammograms at various time scales (Figure 5, S6 and S7). At fast scan rates (2000 mV.s-1), the peak shaped current responses observed for Fc(MeOH)2 (Figure 5A-B and S6A-B) and Ru(NH3)6Cl3 (Figure S7) at both unmodified and polymercoated electrodes corresponds to a quasi-reversible electron transfer and to mass transport by semi-infinite planar diffusion. Under these conditions, the peak currents scale with the concentration and the square root of the diffusion coefficient of the redox probe (Figure S9B and S9E). For the bare electrodes, the peak currents are in excellent agreement with the values predicted from the Randles Sevcik equation (Table S1). The electrodes covered with the polymer film display peak current densities which are about 50 % lower (57 % at 2000 mV.s-1) than the unmodified electrodes (Figure 5C and S6C), indicating that the polymer film decreases the diffusion coeffi-

Figure 5. Effect of polymer film and electrode arrangement on mass transport and redox cycling: (A) CV of the electrode array without polymer film in bulk electrolyte at 2000 mV.s-1 and 2 mV.s-1 (B) Single droplet CV of the electrode array coated with polymer film at 2000 mV.s-1 and 2 mV.s-1. (C) Normalized peak current (ip/ν1/2) vs ν1/2 for an electrode array without polymer using internal (solid black dots) and external counter electrode (open black dots) as well as single droplet on polymer coated electrode array (blue solid dot). The wide, bottom Au bands were contacted as WE. The corresponding CVs for all scan rates are given in Figure S8D-I. All measurements were performed with Fc(MeOH)2 (1 mM) in aqueous KNO3 (1 M).

At slow scan rates (2 mV.s-1), the current response approaches a steady state behavior for the bare electrode (Figure 5A and S6A) and displays a peak shaped current for the polymer-coated electrodes (Figure 5B and S6B). In both cases, the normalized peak currents (ip/ν1/2) are sharply increasing with decreasing scan rates (Figure 5C and S6C). This trend is observed whether a Au band is employed as internal counter electrode (solid black dots, Figure 5C and S6C) or a remote external counter electrode is used (open black dots, Figure 5C


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and S6C) implying that a redox cycling process between the counter and the working electrode19 does not significantly contribute to the current response. Instead, the current increase observed at increasing experimental time scales is attributed to the transition from semi-infinite planar diffusion to hemispherical diffusion. For sufficiently long time scales, the theory of hemispherical diffusion at microband electrodes predicts a quasi-steady state current.41,42 For the uncoated Au bands the quasi-steady state currents for Fc(MeOH)2 (Figure 5A and S6A) are in good agreement with the theoretical values (1.48 µA and 1.27 µA, respectively, at 10 mV.s-1, Table S1) which demonstrates a microband electrode behavior with defined mass transport. In the case of the polymer coated electrode, the dimension of the hydrophilic spots (600  600 µm), and possibly the height of the droplet (in total about 60 µm for the minimal droplet size of 20 nL), prevent the transition to a pure hemispherical diffusion. At 2 mV.s-1, a peak shaped current is obtained whether the wide Au bands (2  200 µm in width, Figure 5B) or the narrow Au bands (72 µm in width, Figure S6B) are contacted as WE which validates the droplet height as being the main contribution to the restricted volume effect. Hence, the droplet boundaries restrict the expansion of the diffusion layer at long time scales which explains the generation of a peak shaped current instead of the quasi-steady state current that would be expected if diffusion remained semi-infinite.

Figure 6. Droplet microarray with binary composition containing hexacyanoferrate (red) or electrolyte only (blue) (A) Optical microscope image of 3  3 single droplets (600 µm  600 µm, ~20 nL each) on the electrode microarray (B) CVs of 3  3 single droplets with hexacyanoferrate and electrolyte arranged alternately. The combinations of electrodes used for addressing each single droplet are given in the respective CVs. The hexacyanoferrate concentration is 1 mM and the electrolyte is aqueous KNO3 (1 M). The narrow top Au band was used as WE. Scan rate is 100 mV.s-1.

Individual addressability. Each Au band serves both as wiring and electrode for up to 20 electrochemical cells. Addressing individual droplets within the eDMA requires that the electrochemical process is limited to the droplet at the intersection of the respective CE and WE without interferences from the other droplets in contact with these band electrodes. To verify this, local drop-spotting was used to form a binary array of alternating droplets containing either buffer only or hexacyanoferrate, thus ensuring that each band electrode is exposed to the two types of solutions (Figure 6A). By contacting two band electrodes, the resulting electrochemical response corresponds to the content of the droplet at the intersection of the electrodes. The oxidation and reduction currents for hexacyanoferrate are detected exclusively in the droplet initially containing the redox probe (Figure 6B). The absence of any significant faradaic signals in the droplets containing buffer only (Figure 6B, the center and the 4 corner droplets) demonstrates that the hydrophobic boundaries efficiently block both the ion transport and the diffusion of redox molecules between droplets. Therefore, the individual addressability is ensured and cross-contamination is prevented. Reproducibility, stability and reusability. The narrow Au band (72 µm) and its orthogonally positioned large dual band electrodes (2 × 200 µm) can be used as counter or working electrode interchangeably. In principle, electroanalytical systems rely on oversized counter electrodes to ensure nonlimiting current flow. Nevertheless, for low current densities, the larger band electrodes may also be used as working electrodes, as illustrated by the cyclic voltammograms of Fc(MeOH)2 in a single droplet (Figure S5B). This opens up the possibility to electrochemically access the full droplet footprint. After droplet drying and refilling with distilled water, comparable peak currents are recovered (Figure S5C). This implies that deposition can be performed regardless of droplet evaporation since refilling of the dried droplets can be achieved on the complete array in a single step by water vapor condensation or by drop casting. The droplet to droplet reproducibility of the eDMA was evaluated by testing a total of 28 single droplets containing Fc(MeOH)2 (Figure S10A-B). The relative standard deviations for the oxidation (ipa) and reduction peak currents (ipc) are 9.4% and 22.1% respectively. The ipa and ipc fluctuations are accounted for the partial evaporation of the droplets as well as variations in the electrode sensitive area or diffusion variability. The standard deviation in formal redox potential (E0’) from droplet to droplet is only 14 mV (n = 28). The stability of E0’, ipa and ipc was also monitored in a single droplet for 100 cycles at 50 mV.s-1 in presence of Fc(MeOH)2 (Figure S11A-B). While the baseline current shifted by about 15 nA, the current amplitude (ipc - ipa) fluctuated by less than 1.3 %. E0’ decreased by only 16 mV. It should be mentioned that the presence of the Fc(MeOH)2, as a fast redox couple in the electrolyte, contributes to the reasonable stability of the potential of the pseudo-RE. More generally, when using a Au band electrode as pseudo-RE, the potential must be checked, possibly by adding a reference electroactive compound. Alternatively, the bare Au bands can in principle be converted into a true reference electrode by electrodeposition of Ag/AgCl in order to achieve a more stable reference potential in all types of experiments43.



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In addition, both the peak currents (ipa = 105 +/- 11 nA and ipc = -79 +/- 13 nA) and E0’ (47 +/- 8 mV vs Au pseudo RE) obtained for Fc(MeOH)2 are mostly unaffected (slightly increasing ipa and ipc) upon washing and re-filling a single droplet with a fresh solution containing the redox species (Figure S11C,D). Therefore, the eDMA can be recycled at least five times. Electrochemical sensing in single microdroplets. In the eDMA system, the surface area of the top narrow Au band within a droplet is 4.32  10-4 cm2 and its ratio to droplet volume (20 nL) is 2.16  10-5 cm-1. Hence, for continuous electroanalytical application, the use of this Au band as working electrode will minimize the electrochemical consumption of species within the droplets. In contrast, if the wide Au bands are used as working electrodes, the working area is 1.67  10-3 cm2 and its ratio to volume is 8.48  10-5 cm-1. Hence using the wide Au electrode could also be useful for electrolysis experiments when full conversion of irreversible redox species is targeted. High-throughput screening of living cells by electrochemical read-out will rely on the sensing of redox-active reporter molecules used in cell activity assay such as quinones44 or of molecules consumed or released by living cells, such as H2O2.45 We select para-benzoquinone as a chemically reversible analyte to test the eDMA for quantification purpose by means of voltammetric methods. The CVs of the quinone within single droplets were recorded at concentrations ranging from 0.5 to 25 mM (Figure 7A). The peak separation between anodic and cathodic currents (ipa and ipc, respectively) is significantly larger compared to the one observed on freshly polished Au electrodes (Figure S12) and is indicative of a quasireversible electrochemical behavior attributed to the relatively complex electrochemistry of quinones.46 Nevertheless, quantification within the individual droplets is possible as demonstrated by the good sensitivity (6.5+/-0.9 nA mM-1 for ipa and 10.9 +/-0.3 nA mM-1 for ipc) extracted from the peak currents (Figure 7B) and by the low relative standard deviation obtained for repetitions in three different droplets for each concentration (25.5 % for ipa and 8.5 % for ipc on average). In addition, we select H2O2 as an example for an electrochemically irreversible analyte. Again, H2O2 oxidation on the polymer-coated Au electrode show significant overpotential (Figure 7C). Nevertheless, quantification of H2O2 is possible within the single droplets as demonstrated by the reasonable standard deviation and sensitivity (1.20+/-0.2 nA mM-1) extracted from the calibration curve (Figure 7D). The sensing performances in terms of detection limit and overpotential of the polymer coated Au band electrode are expected to be significantly improved through surface modification of the band electrodes serving as the electroanalytical probe.47–50 Moreover, for most analytical targets, chronoamperometry is often preferred over cyclic voltammetry owing to its simplicity of use. To evaluate the possibility of amperometric detection within single droplets, we compare the mass transport parameters (D1/2·C) of Fc(MeOH)2 obtained from CV and from chronoamperometry (Figure S13A-D). The D1/2·C value extracted from fast scan rates CV (1000 mV.s-1) is 2.1  10-9 ± 2  10-10 mol.cm-2.s-1/2 which is in reasonable agreement with the value of 3.0  10-9 ± 6  10-10 mol.cm-2.s1/2 obtained from chronoamperometry at short times (0.4 to 20 s). A similar agreement is found for the apparent steady-

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state current (22 ± 1 nA) obtained for slow scan rate CV (1 mV.s-1) and the one observed (19 ± 1 nA) in chronoamperometry after 120 s. Hence the eDMA are in principle suitable for amperometric detection in single droplets.

Figure 7. Electrochemical sensing of 1,4-benzoquinone and H2O2 within single droplets. (A) CVs of 1,4-benzoquinone for concentration ranging from 0.5 mM to 25 mM. (B) Calibration curve of anodic peak current for 1,4-benzoquinol oxidation (solid black dots) and of the cathodic peak current for reduction of 1,4benzoquinone (solid black squares) vs. the concentration. The standard deviation is obtained from three repetitions in different droplets for each concentration. (C) CVs of H2O2 for concentration ranging from 1 mM to 50 mM. (D) Calibration curve of the maximum current for H2O2 oxidation vs. the H2O2 concentration (solid black dots). The standard deviation is obtained from three repetitions in a single droplet washed and reused for each concentration. The electrolyte is KNO3 (1 M). The narrow top Au band was used as WE. The scan rate is 100 mV.s-1.

CONCLUSION The microdroplets with integrated electrodes form an array of electrochemical cells that meet the requirements of standard electrochemical methods such as cyclic voltammetry and chronoamperometry. The cross-bar arrangement of the band electrodes makes it possible to individually address up to 400 droplets with only 41 contact pads (20 WE, 20 CE and 1 pseudo-RE) on the eDMA. The porous polymer layer covering the underlying electrodes is fully permeable to the electrolyte and the electrodes display the expected electrochemical behavior of Au. The diffusion of redox active analytes through the hydrophilic parts of the polymer film is possible irrespective of their charge and their electrochemical detection and quantification is possible irrespective of their electrochemical reversibility thus demonstrating the wide applicability of the eDMA


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platform. At short experimental time scales, the single droplet electrochemical cells containing reversible redox couples essentially behave as standard three-electrode cells under semi-infinite diffusion. At longer time scale, the diffusion layer thickness associated to the electrochemical processes reaches the droplet boundaries which results in a modified quasi-steady-state current. The pattern of hydrophilic spots on a highly hydrophobic surface leads to the effect of discontinuous dewetting and makes it possible to create hundreds to thousands of completely separated nanoliter-sized droplets in a single step. The spatial separation of the microdroplets through the air-filled hydrophobic porous polymer blocks ion transport and analyte diffusion, ensuring both individual electrochemical addressability and absence of cross-contamination. These advantages combined with the reproducibility and reusability of the system make the eDMA valuable as an electrochemical read-out for various screening applications, for example, biochemical or living cell screens. In principle, any analyte for which an electrochemical sensing concept was developed can now be transposed to the eDMA by using adequate band electrode materials/surface modification to monitor the cell activity within the droplet. In addition, the conceptual design of the eDMA based on three-electrode systems opens the possibility for operation in an actuator-sensor mode, whereby a timecontrolled electrolytic process may be exploited to form concentration gradients across the droplet array. Electrochemical activation of components of the solution such as drugs or electrochemically induced pH changes will serve as tools to study cell behavior. Finally, the eDMA platform will be transposable to various active biological materials such as proteins and DNA or even to inorganic systems such as nanoparticles.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental procedure for fabrication of the eDMA and associated flow chart; droplet formation on the eDMA; electrochemical behavior of the Au electrodes in electrolyte only and in presence of Fc(MeOH)2 and Ru(NH3)6Cl3 at various scan rates; reproducibility, stability and reusability of the eDMA, comparison of experimental and calculated current values; CVs of Au disk electrode in presence of benzoquinone; comparison of chronoamperometry and cyclic voltammetry (PDF).

AUTHOR INFORMATION Corresponding Author * [email protected] * [email protected] ORCID Huijie Zhang: 0000-0001-8149-8637 En Ning Saw: 0000-0003-1907-5611 Nicolas Plumeré: 0000-0002-5303-7865

Author Contributions PL and NP conceived the study. TA, TO and AL designed and fabricated the band electrode microarray. WF and PL coated and patterned the eDMA. ENS and TA performed initial characterization of the eDMA. HZ performed in-depth, full characterization of the eDMA and single droplet studies. HZ, AL, PL and NP wrote the manuscript.

Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft (DFG) and China Scholarship Council studentships to H. Z. and W.F. P.L. and W.F. were supported by the ERC Starting Grant (ID:337077-DropCellArray), ERC PoC (ID: DLV-680913CellScreenChip) and the Helmholtz Association's Initiative and Networking Fund (Grant No. VH-NG-621). We would also like to thank Dr. Philippe Fortgang for preliminary investigations and valuable discussions and Ivana Pini for the help with SEM investigations, polymer coating and contact angle measurements.

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High-Density Droplet Microarray of Individually Addressable

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