Nanoscale Electrochemical Sensor Arrays: Redox Cycling

Article pubs.acs.org/accounts

Nanoscale Electrochemical Sensor Arrays: Redox Cycling Amplification in Dual-Electrode Systems Published as part of the Accounts of Chemical Research special issue “Nanoelectrochemistry”. Bernhard Wolfrum,*,†,‡ Enno Kaẗ elhön,†,§ Alexey Yakushenko,† Kay J. Krause,† Nouran Adly,† Martin Hüske,†,∥ and Philipp Rinklin‡ †

Institute of Bioelectronics (PGI-8/ICS-8), Forschungszentrum Jülich, 52425 Jülich, Germany Neuroelectronics, IMETUM, Department of Electrical and Computer Engineering, Technical University of Munich, Boltzmannstr. 11, 85748 Garching, Germany

‡

S Supporting Information *

CONSPECTUS: Micro- and nanofabriation technologies have a tremendous potential for the development of powerful sensor array platforms for electrochemical detection. The ability to integrate electrochemical sensor arrays with microfluidic devices nowadays provides possibilities for advanced lab-on-a-chip technology for the detection or quantification of multiple targets in a high-throughput approach. In particular, this is interesting for applications outside of analytical laboratories, such as point-of-care (POC) or on-site water screening where cost, measurement time, and the size of individual sensor devices are important factors to be considered. In addition, electrochemical sensor arrays can monitor biological processes in emerging cell-analysis platforms. Here, recent progress in the design of disease model systems and organ-on-a-chip technologies still needs to be matched by appropriate functionalities for application of external stimuli and read-out of cellular activity in long-term experiments. Preferably, data can be gathered not only at a singular location but at different spatial scales across a whole cell network, calling for new sensor array technologies. In this Account, we describe the evolution of chip-based nanoscale electrochemical sensor arrays, which have been developed and investigated in our group. Focusing on design and fabrication strategies that facilitate applications for the investigation of cellular networks, we emphasize the sensing of redox-active neurotransmitters on a chip. To this end, we address the impact of the device architecture on sensitivity, selectivity as well as on spatial and temporal resolution. Specifically, we highlight recent work on redox-cycling concepts using nanocavity sensor arrays, which provide an efficient amplification strategy for spatiotemporal detection of redox-active molecules. As redox-cycling electrochemistry critically depends on the ability to miniaturize and integrate closely spaced electrode systems, the fabrication of suitable nanoscale devices is of utmost importance for the development of this advanced sensor technology. Here, we address current challenges and limitations, which are associated with different redox cycling sensor array concepts and fabrication approaches. State-of-the-art micro- and nanofabrication technologies based on optical and electron-beam lithography allow precise control of the device layout and have led to a new generation of electrochemical sensor architectures for highly sensitive detection. Yet, these approaches are often expensive and limited to clean-room compatible materials. In consequence, they lack possibilities for upscaling to high-throughput fabrication at moderate costs. In this respect, self-assembly techniques can open new routes for electrochemical sensor design. This is true in particular for nanoporous redox cycling sensor arrays that have been developed in recent years and provide interesting alternatives to clean-room fabricated nanofluidic redox cycling devices. We conclude this Account with a discussion of emerging fabrication technologies based on printed electronics that we believe have the potential of transforming current redox cycling concepts from laboratory tools for fundamental studies and proof-ofprinciple analytical demonstrations into high-throughput devices for rapid screening applications.

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INTRODUCTION Stimulating and recording signals from biological cell-networks is of fundamental interest for applications ranging from neuroprosthetics, to replace lost functionality, to cell-based biohybrids for disease modeling and screening of drug effects. Consequently, years of research have been invested in developing functional interfaces that can transduce information © XXXX American Chemical Society

between cellular and artificial systems. In this regard, systems that record or stimulate electrical signals of cells, e.g., action potentials, are already quite advanced, with modern architectures currently comprising thousands of recording sites.1,2 Received: June 30, 2016

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DOI: 10.1021/acs.accounts.6b00333 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research

Figure 1. (a) Schematic of on-chip detection of vesicular neurotransmitter release. (b) Amperometric traces recorded at multiple channels during vesicular dopamine release from PC-12 cells, scale bar 50 pA. (c) Magnification of a single spike recorded from a rat midbrain neuron growing 21 days on-chip. (d) SEM images of PC-12 cells growing on microelectrode arrays. White arrows indicate cells, yellow arrows indicate the microelectrode, scale bars: 10, 5, 2.5, and 1 μm (i-iv, respectively). Partially adapted from ref 9. Copyright 2013 American Chemical Society.

capacitively coupled voltage signals.9,10 The general concept of neurotransmitter detection from cells on-chip and corresponding recordings are shown in Figure 1. Neurotransmitters are released from vesicles at the cell membrane and diffuse to individual sensor spots. The released molecules are then oxidized at an appropriately biased electrode and generate a current spike. The integral of this current spike yields the amount of transferred charge and is indicative of the number of neurotransmitters that was stored within the vesicle. Due to the low number of molecules that are released from each vesicle, the generated signals are rather small (Figure 1c). As a consequence, it is usually only possible to detect vesicular release directly at or within a few micrometers of the sensor and many signals in a network will be lost due to diffusional dilution.9 Further problems are associated with possible interference of molecules (e.g., ascorbic acid) that can be oxidized at similar potentials as the molecule of interest. The challenge of detecting minute electrochemical signals of a target analyte in a, sometimes unknown, background is not only encountered in the investigation of neurochemical release but represents in fact a major obstacle for the development of electrochemical sensor technology for biological and medical applications. Beyond cell measurements, various electrochemical techniques have been applied for detection of biological molecules such as antigens in immunoassays, peptides, DNA or RNA. These can be either measured directly, if they are inherently electrochemically active, via a conjugated electrochemical label, or indirectly with an electrochemical

However, in contrast to purely electrical methods, techniques for recording the chemical activity of cellular networks in a high-throughput approach are still rather limited. In neurochemical investigations, it is desirable to monitor neurotransmitter levels with high spatial and temporal resolution. Conventionally, these investigations are carried out with probe-based methods using carbon-fiber electrodes.3−6 Such electrodes can be easily produced in the laboratory and provide a flexible method for manual handling in standard cell culture. However, their lack of high-throughput capabilities severely limits these approaches for studies on a cell-network scale. Furthermore, in recent years there has been a tremendous progress in cell-culture model systems in microfluidic devices, which are difficult to access with probe-based techniques. Importantly, microfluidic cell-culture systems, mimicking specific neural pathways, have great potential for investigating disease models and would strongly benefit from the possibility to record real-time neurochemical activity during long-term experiments.7 For example, monitoring chemical communication in reconstructed nigrostriatal pathways on-chip would allow the investigation of progressive neurodegeneration at a functional level. A possibility to combine microfluidic cellculture systems with functional recording units is given by integrating electrochemical sensor arrays directly into the substrate.8 This is similar to the application of microelectrode arrays for action potential recordings in cell-networks, although in this case the signals are generated by the Faradaic reactions of neurotransmitters at the microelectrodes rather than B


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Accounts of Chemical Research mediator.11 A major goal for all of these applications is to achieve a selective signal amplification through the choice of an appropriate sensing mechanism.

An alternative approach, which does not require highresolution lateral fabrication, relies on the deposition of individual electrode layers on top of each other. Specifically, a thin physical-vapor-deposited chromium film acts as a spacer between two gold or platinum electrodes. In this configuration, the electrode separation can be precisely tuned by the thickness of the deposited chromium layer.16 In a subsequent step, the sacrificial layer is dissolved leaving a gap between the electrodes. In the past decade, the Lemay group has pioneered redox-cycling sensors using this technology and fabricated vertically stacked electrodes within nanofluidic channels of only few tens of nanometers in height. The development of nanofluidic redox-cycling sensors has been driven by the desire to answer fundamental questions in electrochemistry and, in particular, to demonstrate the ultimate level of single-molecule detection in a reliable and scalable microfabricated device.17−20 Due to the defined geometric arrangement of the electrodes, it is straightforward to calculate the electrochemical current that is generated by a certain analyte concentration or number of analyte molecules within a nanofluidic sensor. For experiments performed in solutions comprising supporting electrolytes (typically larger than 100 mM), electrophoretic effects are screened effectively within the nanometer range at the electrodes and only have a minor influence if the distance between the electrodes is above ∼50 nm. Neglecting kinetic limitations, we can estimate the redox-cycling current irc for a fixed concentration c or number of molecules n in the confined region between the electrodes

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ON-CHIP REDOX CYCLING A highly attractive amplification concept, which virtually calls for miniaturization of devices on the nanoscale, is the electrochemical redox-cycling principle. Similar to the concept of environmental recycling, the technique relies on converting a “waste” material into a reusable product. In single-potential amperometric measurements, the waste product is given by a redox molecule, which becomes useless for further detection after the reaction at the working electrode. If, however, a nearby second electrode regenerates the molecule to its original state, it is recycled and can react again with the working electrode. Overall, the molecule will transfer charges during every successive reaction, effectively amplifying the detected current per molecule. The redox-cycling concept can be realized by biasing two closely spaced electrodes to a potential above and below the redox potential of the molecule. Fan and Bard have applied this method to detect the current of individual molecules that were confined in the space between a scanning electrochemical microscopy tip and a conducting surface.12 The recycling method can greatly amplify electrochemical current signals for the detection of molecules if the time between oxidation and reduction reaction is short. In turn, molecules contribute with many individual electron transfer events per unit time generating a large current that can be detected by appropriate amplification circuitry. The time for the repetitive electron transfer itself depends on the distance the molecule has to travel between the electrodes. Generating efficient redox-cycling amplification on a chip therefore calls for technologies that permit the fabrication of electrode pairs separated only by a very small gap. With the rise of thin-film technology, microfabrication has become a powerful tool for the fabrication of substrate-based redox-cycling electrodes.13,14 In particular, micrometer-scaled interdigitated electrodes (Figure 2) were fabricated using robust and scalable processes

irc =

zFDA zeD c= 2 n h h

Here the diffusion coefficient D is typically on the order of 10−9m2 s−1, F = 96 485 C/mol is the Faraday constant, −e = −1.6 × 10−19 C is the electron charge, A is the area of electrode overlap, and z is the number of electrons transferred per molecule and cycle. A molecule crosses the gap twice during each cycle and, for a divalent reaction (z = 2) and an electrode distance of h = 100 nm, the current amounts to i = 32 fA, although realistic values are lower in particular due to kinetic limitations or reversible molecular adsorption.18,20−23

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REDOX-CYCLING AMPLIFICATION The amplification of redox-cycling devices can be assessed for different applications and several definitions of the amplification factor have been reported in literature. The most common definition is simply given by the ratio of currents obtained when using the same device in redox-cycling mode irc and nonredox cycling mode inrc (meaning that only one of the working electrodes is active for either oxidation or reduction): Γsel =

irc inrc

This definition is useful if we ask the question: To what extent does the device amplify the current caused by target molecules that can participate in redox cycling vs the current that is observed solely due to the diffusive mass transport from the bulk to one of the electrodes? It is particularly useful if signals from one analyte are to be selectively amplified against a background signal caused by molecules that do not participate in redox cycling or interact with the target. For example, this applies if we want to observe a catechol-based neurotransmitter, such as dopamine, in the presence of ascorbic acid.16

Figure 2. Schematic of redox cycling at an interdigitated array.

based on optical lithography and lift-off or reactive ion-etching techniques. However, these devices rely on lateral structuring, which is limited by the resolution of the fabrication method.15 A transition from micro- to nanoscale devices is therefore inherently difficult and requires advanced and low-throughput fabrication technologies such as electron-beam fabrication. C


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Figure 3. (a,b) Schematic of a nanocavity redox-cycling array. (c) SEM image of a focused-ion-beam-induced (FIB) crosssection through a nanocavity sensor (scale bar 150 nm). (d) Comparison of different sensor responses to a local stimulus calculated using random walk simulations.

associated with the application and it is therefore useful to take a closer look at several properties of redox cycling in nanoscale geometries. Naturally, the system requires a redox-active target or tracer. In terms of the lower “limit of detection”, nanofluidic redox-cycling devices with single-molecule resolution have in theory the possibility of detecting any concentration of redoxactive targets, if the experimentalist is willing to invest enough time. Since the frequency of detected events relates linearly to the bulk concentration, the recording time directly correlates with the sensitivity. The caveat is, however, that at extremely low concentrations we might have to wait long until a single molecule enters the sensor and triggers a detectable signal. For experiments conducted with static bulk concentrations of the analyte, we can calculate the probability of a single molecule residing within the active amplification volume of the nanofluidic sensor. For example, a typical nanofluidic redox cycling sensor with a length, width, and height of 100 μm, 2 μm, and 100 nm, respectively, coupled to a bulk reservoir with an analyte concentration of 100 fM contains on average 0.0012 molecules within the sensor. It is evident that depending on the geometric layout and associated residence times of the molecules within the sensor, experimental time scales will get very long. However, typical sensor applications, such as wastewater screening or point-of-care analysis require signals to be obtained within a reasonable time of seconds or minutes. Even more critical is the application of nanofluidic redoxcycling sensors for the detection of spatiotemporal signals, such as vesicular release of neurotransmitters within a cell network. Here, in addition to the temporal resolution a high spatial resolution on the μm-scale is required for identifying release events by individual cells.

In general, the definition above is not useful if we are interested in comparing the current that can be generated by different devices at a certain concentration. For redox-cycling devices in nanofluidic channels, inrc tends to be very small due to the restricted access to the bulk medium. With the definition above, this will grant a high amplification factor without necessarily providing a high current amplification compared to amperometric sensors of similar size.24 If we are interested in designing sensor arrays, it is therefore more useful to relate the redox-cycling current to the current of an established system. In this respect, a planar circular microelectrode provides a good reference value as it can be easily fabricated on a chip. We can readily provide an expression for this theoretical amplification factor, assuming diffusion-limited steady-state current ime of a chip-based microelectrode: Γsen =

irc A ∼ 4hr ime

From this relation we directly get an impression how the amplification scales for a particular sensor geometry. As the redox-cycling current depends on the area of electrode overlap but the steady-state current of a single microelectrode just scales with the radius r, it is clear that we get the largest redoxcycling amplification for laterally extended devices with small electrode distances h.

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SPATIAL AND TEMPORAL RESOLUTION Redox-cycling exhibits a powerful current amplification scheme, yet the question arises: Can we employ nanofluidic redoxcycling devices for analytical sensing applications? The answer of course depends on the specific requirements that are D


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the effect of redox-cycling amplification becomes apparent. Both redox-cycling architectures, nanocavity and nanofluidic channel, exhibit significant signal amplification compared to the simple amperometric microelectrode recording. The maximum current for the nanocavity sensor is higher than for the nanofluidic channel. This is readily explained by the central position of the aperture within the circular nanocavity, which allows more molecules to access the zone of redox-cycling amplification before escaping into the bulk. Molecules, which have managed to enter the nanofluidic redox-cycling channel have a lower chance of escaping back into the bulk solution, leading to a longer tail and slower signal component. Overall, when designing redox-cycling devices for sensing applications, care has to be taken with consideration of the spatiotemporal requirements. While long channel-like structures with high redox-cycling efficiencies are ideally suited to resolve fluctuations of individual molecules, circular nanocavity electrodes are beneficial for resolving faster time-dependent phenomena.

While nanofluidic redox-cycling devices have a tremendous potential for single-molecule experiments, they are rather limited in terms of temporal resolution. This is directly understandable as a particle that is located within a channel of several hundred μm in length, typically exhibits a long residence time due to the square-root dependence between time and space for diffusion-driven processes. In fact, for single-molecule experiments this long residence time is a crucial requirement as the fA-signal generated by an individual molecule cannot be recorded at a high bandwidth due to noise limitations. It is therefore important that the molecules stay for an extended time, typically on the order of seconds, within the sensor to generate an adequate signal to be detected. The spatial resolution of a chip-based redox-cycling sensor is simply determined by the geometry of the access apertures of the sensor. Thus, any channel-like design is unfavorable since molecules can enter via two spatially separated apertures, which are typically several tens or hundreds of μm apart. The problem of limited spatial resolution can be easily addressed by removing one access of the nanofluidic redox-cycling channel, effectively turning the channel into a cavity device. To avoid problems of temporal resolution that arise due to long residence times of molecules inside an elongated channel structure, the access aperture for the redox-cycling sensor can be set centrally above the nanocavity.25,26 A circular-shaped cavity ensures that the maximal distance between access hole and sensor volume is minimized leading to a faster response time. We have introduced this design in a microelectrode array, a standard tool for investigating electrical activity of neuronal cell cultures. Individual sensors in this array have two vertically stacked plane-parallel circular electrodes that are separated by a nanoscaled gap (Figure 3a−c). The fabrication of these devices solely relies on standard optical lithography with sacrificial layer etching, giving rise to opportunities for parallel clean-room processing. An array of these sensors allows spatiotemporal mapping of concentration fluctuations. We have demonstrated this by resolving fluctuations of a dopamine concentration gradient within a microfluidic channel.25 At the same time, the devices are particularly useful for the direct detection or stimulation of electrophysiological activity from cells. Here, the increased electrode−electrolyte interface within the nanocavity in combination with a small sensing aperture gives rise to highly localized and efficient cell-chip coupling conditions for both recording27 and stimulation28 of action potentials at individuals cells of a network growing on the chip surface. In general, the spatiotemporal response of nanocavity sensor arrays may be complex depending on the exact geometric constraints and related boundary conditions, rendering an analytical description difficult. Thus, numerical solutions, for example based on finite-element methods or random walk simulations, have to be employed to describe the response to certain stimuli. In particular, random walk simulations are useful as they reflect the discrete nature of the analyte molecules and allow insight into sensors’ noise characteristics and processes on the molecular scale. Figure 3d shows the simulated response of a simple microelectrode, a circular nanocavity redox-cycling sensor and a nanofluidic channel to an instantaneous point-like release of 10 000 molecules located centrally above the sensor’s aperture. The geometric arrangement models a release site of a neurotransmitter-containing vesicle. A detailed description of the sensor layouts is given in the Supporting Information. Comparing the sensor responses,

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REDOX CYCLING CROSS BAR ARRAYS Another important aspect to consider in the design of redoxcycling sensor arrays is the sensor density. Being able to integrate many sensors in an array is highly desirable for monitoring neurochemical communication in cell networks and the detection of localized gradients or multiple targets. Due to the large signal amplification, sensor devices themselves can be fabricated rather small, typically in the range of several μm. The limiting factor in this respect is the integration of feedlines, in particular when advanced processes for feedline fabrication are avoided to reduce fabrication cost and time. Intuitively one might argue that the fabrication of dense redox-cycling sensor arrays is more complicated in comparison to standard microelectrode arrays by the fact that at least two feedlines instead of one are needed to fabricate a single sensor - one for the reducing and one for the oxidizing electrode. However, this issue can be resolved by arranging the feed lines in two orthogonal arrays where the electrochemical redox-cycling sensors are located at each intersection of the feed lines. Individual sensors are then addressed by biasing two perpendicular feed lines to appropriate potentials below and above the redox potential of the target molecule. Such addressable redox-cycling sensor arrays have been introduced and employed by the group of Matsue since 2008 using different geometric layouts29 including interdigitated electrode designs. Here, the number of sensors scales with the square of the number of feedlines (n2) and thus allows a significantly higher sensor density. Due to the strong redox-cycling amplification, this concept is also highly interesting for the development of nanocavity sensor arrays. We have designed a sensor array with 256 sensors and 32 feedlines by integrating the nanocavities in a crossbar array (Figure 4).30 As the sensors for nanocavity devices are directly integrated between the feedlines no extra space for the sensor fabrication at the intersection is required. Here, the strong signal amplification and efficiency of the redox-cycling process are of major benefit. While Faradaic currents can arise at all sensors that are connected to one of the activated feed lines, their contribution will be small compared to the amplified redox-cycling signal. This way, individual sensors can be read-out without major interference from other sensors. In fact, since nanocavity redoxcycling devices exhibit an almost perfect anticorrelation of cathodic and anodic currents, it is possible to operate a section E


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address these strategies and describe the current status in nextgeneration redox-cycling devices based on nanoporous sensor arrays.

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NANOPOROUS SENSOR ARRAYS An elegant approach to counter stability issues in nanoscale redox-cycling devices without compromising the advantages of vertically stacked electrode systems is the introduction of porous electrode and insulator layers.32−34 Devices based on this concept exhibit a porous top electrode that faces the electrolyte solution. The bottom electrode required for redoxcycling connects to the bulk via individual pores that penetrate both, the top electrode and the thin insulating layer between the electrodes (Figure 5).

Figure 4. Nanocavity redox-cycling cross-bar array. (a) Schematic representation, (b) microscopic image (scale bar: 50 μm).

or the whole sensor array in a parallel readout mode by activating all feedlines at once. In general, a continuous concentration distribution will then deliver current traces that cannot be mapped unambiguously to individual sensor locations. However, for the detection of sparsely distributed spatiotemporal signals, such as the detection of neurotransmitter release, the recorded signals can be attributed to the sensors by cross-correlating the signals from oxidizing and reducing electrodes. Overall, this allows a faster readout of signals and avoids unwanted interference that arises due to the switching of the electrode potentials. While current cavity-based redox-cycling devices have demonstrated great potential for fundamental electrochemical sensing techniques,25,31 several issues still have to be addressed to improve their usability in a broader range of applications. One of the main issues concerns the fabrication process and stability of devices. The nanocavity design, leaving a tiny gap between top and bottom electrodes, poses a risk for failure if the cavity collapses and produces a short contact. This is particularly dramatic in the crossbar array layout where a single failure leads to the destruction of a whole row and column (n− 1 sensors). Furthermore, due to the strongly confined cavity, sensors may exhibit a rather high failure rate for repeated application, in particular if cleaning protocols have to be applied. There are basically two strategies to counter this challenge: Obviously, one route is to develop nanoscale redoxcycling designs, which do not, or to a lesser extent, suffer from stability issues. The other option is the development of a fabrication process that is so fast and cheap that sensor arrays can be produced in a high-throughput manner as disposable devices without significant cost. In the following, we will

Figure 5. Schematic (a) and SEM top (b) and side view (c) of a nanoporous redox-cycling sensor array. Scale bars for (b) and (c) are 200 and 100 nm, respectively.

While it is straightforward to fabricate the layered stack of electrodes, the question arises how to integrate nanopores within the device. For this task, we have chosen a process based on electron beam lithography in combination with reactive ion etching.35 Our nanoporous redox-cycling devices are made of platinum electrodes that are separated by a silicon nitride insulator layer of 100 nm thickness. The pores feature a typical radius of 40 nm and are ordered in a hexagonal arrangement at an interpore distance of 200 nm, resulting in a pore density of ∼2.9 × 1013 m−2. Individual sensors have a lateral extension of several tens of μm and the largest devices feature up to 209 000 pores. Nanoporous dual-electrode systems combine an easy access of molecules for redox-cycling amplification with the close spacing of electrodes in a vertical alignment. In comparison to the nanocavity design, these sensors therefore typically respond very fast and can monitor concentration fluctuations at high temporal resolution, depending on the amplification circuitry. Typical electrode area-specific sensitivities that we observe with our nanoporous devices lie in the range of 2−3 × 104 A M−1 m−2 with maximum values of 8.1 × 105 A M−1 m−2 for detecting the redox tracer ferrocene dimethanol, Fc(MeOH)2 at the highly constricted nanopore F


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Accounts of Chemical Research Table 1. Comparison of Different Redox-Cycling Sensor Array Architectures electrode spacing [nm]

type 16

nanofluidic (Lemay group) nanocavity25 nanocavity30 (cross bar) nanoporous35 (e-beam) nanoporous41 (alumina) nanoporous (printed)

target

∼55

catechol

∼65 ∼65

no. sensors/ device

no. apertures/ sensor

fabrication method

area-specific sensitivity [AM−1 m−2]

Γsen

∼1.1 × 10

6

∼34

3

2

e-beam

dopamine Fc(MeOH)2

30 256

1 1

optical optical

∼0.9 × 105 ∼(2−17) × 104

∼8 ∼0.2−1.5

∼120

Fc(MeOH)2

32

∼105

e-beam

∼(2−81) × 104

∼16−46

∼370

[Fe(CN)6]3−/4−

1

∼109

porous alumina inkjet printing

∼1200

Fc(MeOH)2

25

∼3.6 × 104

∼730

∼5.9 × 103

∼1.2

Γsel

electrode area [m2]

∼10

3

∼8.0 × 10−11 ∼6.4 × 10−10 ∼1.7 × 10−12

∼500

∼(1.2−63) × 10−10 ∼9.0 × 10−6

∼30

∼8.8 × 10−10

Figure 6. Printed redox cycling sensor array. Schematic overview of the printing process (a) and a sensor crosssection (b). (c) SEM image of a FIB cut of the printed sensor layers (scale bar: 200 nm). (d) Exemplary cyclic voltammetry traces recorded with a printed redox cycling sensor (500 μM Fc(MeOH)2 at 20 mV/s).

bottom electrodes. A comparison of different parameters measured with diverse redox-cycling sensor concepts is given in Table 1. Due to the short residence time of individual molecules within nanoporous redox-cycling devices they are not useful for single-molecule detection. However, they provide a good alternative if fluctuating concentration gradients are the target of investigation. Furthermore, their specific geometry, featuring different top and bottom electrodes, makes it possible to probe asymmetric transfer coefficients, which are difficult to determine with standard nanocavity devices. Maybe most importantly for the development of future sensor concepts: Nanoporous devices are particularly suited to investigate specific binding events. In such a scenario, redox-active molecules can act as tracers for the detection. A specific binding event, caused for example by the reaction of an antigen with an antibody or aptamer, causes a blocking of the electrochemical redox-cycling current. For an efficient blocking, these applications require pore diameters, which are on the same order of magnitude as the size of the molecules that are to be detected. Interestingly, not only the signal amplitude but also the signal fluctuations and the corresponding autocorre-

lation or power spectrum is affected by this process. This provides a second set of parameters, which can be exploited for the detection of specific binding events.36 In general, if nanoporous redox-cycling concepts are to be applied in a broader sense, cheaper and faster fabrication technologies than electron-beam lithography must be developed. The group of Bohn has recently presented several studies on nanoporous devices fabricated using nanosphere lithography in combination with multistep lithography.37−40 This is a highly interesting technique as it allows parallel instead of serial processing of devices. We have used a different approach for upscaling the fabrication of nanoporous redox-cycling devices on a 4″-wafer platform making use of a self-organized anodized alumina film as an interelectrode spacer.41 This way we were able to integrate approximately 1 billion pores in a large-area sensor of ∼9 mm2. The interpore distance and pore diameters lie in the range of 100 and 40 nm, respectively and are tunable by the anodization parameters of the fabrication process. These large-scale devices exhibit a current response of up to 330 mA M−1 for the redox-active probe [Fe(CN)6]3−/4− corresponding to a redox-cycling amplification factor Γsen ∼ 730. The high sensitivity and short response time of nanoporous sensors hold G



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great potential especially for real-time measurements using high sensor densities.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

OUTLOOK

Present Addresses §

For future sensor applications, including possibly immunoassays in physiological solutions or wastewater screening, the question arises whether nanoscale redox-cycling devices can be built in a reliable process at large quantities and moderate costs.42−44 While the previously mentioned approaches on selfassembled porous layers are a first step toward upscaling fabrication of nanoscale redox-cycling devices, they still rely on rather costly thin-film deposition techniques and are limited with regard to material choice and flexibility of the fabrication process. In this respect, multilayer printed electronics by additive manufacturing such as inkjet technology is emerging as a highly interesting alternative to classical micro- or nanofabrication. The main advantage of this technology lies in the rapid prototyping capabilities and fabrication, which can possibly operate in a roll-to-roll configuration. In addition, costs are drastically reduced as materials only need to be deposited at the desired location and lift-off technologies become obsolete. However, the limiting factor of inkjet printing is the lateral resolution, which depends on the smallest drop size and typically lies in the range of 10−20 μm. This generally excludes inkjet printing for device fabrication on the nanoscale. Yet, here the concept of vertically stacked nanoporous redoxcycling sensors becomes a major advantage. Using inkjet printing it is possible to tune the processing parameters for the fabrication of porous electrode layers. Different functional layers can then be readily deposited on top of each other to fabricate nanoporous electrochemical redox-cycling sensor arrays. Figure 6 shows the concept, cross section, and characterization of a printed redox-cycling sensor. The top and bottom electrodes are made of printed porous carbon and gold, respectively and a porous polystyrene layer is printed as a spacer layer. A more detailed description of the sensor is given in the Supporting Information. While printing nanoporous redox-cycling sensors is still in its infancy stage of development it could potentially become a powerful tool for biosensing applications, as biofunctionalized inks can be directly integrated in the fabrication process. For example, the nanoparticle inks can be conjugated with biorecognition elements such as antibodies or aptamers using well-established coupling chemistries. We believe that in the future, combining the printing process of individual biorecognition layers with redoxcycling electrodes will pave the way for a broad range of applications of nanoscale electrochemical sensor arrays with multitarget detection capabilities.

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E.K.: Department of Chemistry, Physical and Theoretical Chemistry Laboratory, Oxford University, South Parks Road, Oxford, OX1 3QZ, United Kingdom. ∥ M.H.: FEV GmbH, 52078 Aachen, Germany. Notes

The authors declare no competing financial interest. Biographies Bernhard Wolfrum received his Ph.D. Physics from University of Göttingen. Since 2015 he is a Professor for Neuroelectronics at Technical University of Munich, Germany. His research interests include printed bioelectronic interfaces and nanoelectrochemical sensor concepts. Enno Kätelhön. received his Ph.D. in Physics from RWTH Aachen University, Germany. He is currently a postdoctoral research associate in the group of Richard Compton at University of Oxford focusing on theoretical and computational analysis of nanoelectrochemical systems. Alexey Yakushenko received his doctoral degree from RWTH Aachen University. He is currently a postdoctoral research fellow at PGI8/ ICS8, Forschungszentrum Jülich, working on printed electronics for sensor development. Kay J. Krause received his M.Sc. in Physics at RWTH Aachen University and is currently pursuing his Ph.D. under the supervision of Bernhard Wolfrum at PGI-8/ICS8, Forschungszentrum Jülich, working on noise analysis and electrochemical detection of nanoparticles from solution. Nouran Adly obtained her M.Sc. in Nanobiotechnology and Bioanalysis at Rovira i Virgili University, Spain. She is currently pursuing her Ph.D. at PGI-8/ICS8, Forschungszentrum Jülich, working on the development of electrochemical sensor arrays using inkjet technology. Martin Hüske received his Ph.D. in physics from RWTH Aachen working at the Institute of Bioelectronics, Forschungszentrum Jülich. He is currently employed as an engineer enhancing e-mobility at FEV GmbH. Philipp Rinklin received his Ph.D. from RWTH Aachen working at the Institute of Bioelectronics, Forschungszentrum Jülich. He is currently a postdoctoral research fellow focussing on printed electronics and functional interfaces in the Neuroelectronics Group at Technical University of Munich.

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ACKNOWLEDGMENTS We thank all partners internal (PGI-8/ICS-8) and external who have contributed to the work on nanoelectrochemical sensor arrays, in particular Serge Lemay and Andreas Offenhäusser and their respective research groups. Funding has been provided by the Helmholtz Young Investigator Program (VH-NG-515), The Bernstein Center of Computational Neuroscience (01GQ1004A, BMBF), and the DFG large infrastructure program (INST95/1331-1FUGG).

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.accounts.6b00333. Temporal response for different redox-cycling sensor geometries and time-dependent amplification factor; description of printed redox cycling sensor (PDF)

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Nanoscale Electrochemical Sensor Arrays: Redox Cycling

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