New Frontiers and Challenges for Single-Cell Electrochemical Analysis

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New Frontiers and Challenges for Single-Cell Electrochemical Analysis Jingjing Zhang, Junyu Zhou, Rongrong Pan, Dechen Jiang, James D. Burgess, and Hong-Yuan Chen ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.7b00711 • Publication Date (Web): 25 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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New Frontiers and Challenges for Single-Cell Electrochemical Analysis

Jingjing Zhang1, Junyu Zhou1, Rongrong Pan1, Dechen Jiang1*, James D Burgess2, Hong-Yuan Chen1

1. The State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Jiangsu 210093, China 2. Department of Medical Laboratory, Imaging, and Radiologic Sciences, College of Allied

Health Sciences, Augusta University, Augusta, GA 30912, USA

Email: [email protected]

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Abstract.

Previous measurements of cell populations might obscure many important cellular

differences, and new strategies for single-cell analyses are urgently needed to re-examine these fundamental biological principles for better diagnosis and treatment of diseases. Electrochemistry is a robust technique for the analysis of single living cells that has the advantages of minor interruption of cellular activity and provides the capability of high spatio-temporal resolution. The achievements of the past thirty years have revealed significant information about the exocytotic events of single cells to elucidate the mechanisms of cellular activity. Currently, the rapid developments of micro/nanofabrication and optoelectronic technologies drive the development of multifunctional electrodes and novel electrochemical approaches with higher resolution for single cells. In this prospective, three new frontiers in this field, namely, electrochemical microscopy, intracellular analysis and single-cell analysis in a biological system (i.e., neocortex and retina), are reviewed. The unique features and remaining challenges of these techniques are discussed.

Keywords: single-cell electrochemical analysis, electrochemical microscopy, intracellular analysis, high spatio-temporal resolution, nanoelectrodes


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Classic biological principles including cell theory, gene theory, evolution, homeostasis, and laws of thermodynamics are developed based on measurements from a population of cells under the assumption that all cells of a particular type are identical. However, recent evidence from the analysis at single cells reveals that individual cells in the same type may have significant differences beyond being diseased or not and so the role of such heterogeneity in tissue behavior is truly unchartered.1,2 Most researchers appreciate now that some basic questions can only be addressed at the single cell level; what is the rate of exocytotic release per region of the cell surface for example or how many molecules are released during a particular event?3,4 Furthermore, these high cellular heterogeneities have been proven to induce crucial consequences for the health and function of the entire cellular population as eluded to above.5,6 As a result, new strategies for single cell analyses are urgent to refine these fundamental biological principles for eventual superior diagnosis and treatment of diseases. Technically, one living mammalian cell has micrometer size and thus low total content from a measurement perspective. Additionally, numerous cellular components exist that have fast responses to stimuli at the cellular level and within the larger context of tissues and networks. Gaining an understanding of “what molecules are where at to particular time” and “the timescale on which molecules translocate between intercellular compartments” at single cells is the key to establish the interdependence of a vast array of operant biochemical pathways.7 Therefore, the measurement approaches need to address detection sensitivity and specificity as well as spatio-temporal resolution so that the description of this complex system can be characterized.8,9 However, the historic analytical challenge that “measurement effects the system” is obviously the primary hurdle in single cell and intracellular analysis.10

The measurement either consumes or

modifies the target analyte thus altering its thermodynamic and/or biological activity. Then reality is that a comprehensive mapping of the interworkings of any cell is simply not possible in the strictest sense. Also, it is known that only a few copies of particular enzyme are within an intercellular compartment such as single mitochondrion.11 Quantifying the number of enzyme molecules present and also the catalytic activity exhibited collectively by the group of enzyme molecules such that a perenzyme activity can be established presents an insurmountable impediment. Further, heterogeneity in activity between the different enzyme copies arising from different immediate environments and ATP binding state for example is yet even more out-of-reach.12 The analytical scientist has hence focused much on the background signal in such measurements or the signal that is quantified relative to the signal arising from the target species or physical quantity of interest. Currently, fluorescence microscopy with a near zero analytical background is the most widely adopted tool for single cell analysis and relies on the spectral response upon recognition of fluorescent probes binding to target molecules.13 The design and synthesis of highly fluorescent


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probes has permitted specific detection of analyte inside the cells at the single molecule level.14 Most recently, super-resolution fluorescence microscopies have circumvented the limit of light diffraction to provide the image of molecules with subcellular spatial resolution of < 10 nm.15-17 This revolutionary breakthrough permits the unprecedented visualization of molecules in the subcellular compartment.

However, the perturbation of cellular states after staining of living cells using

fluorescence probes is unknown as is the original influence of the probe on the activity of the target. The temporal and spatial resolution in fluorescence microscopy is often compromised for detailed analysis of intracellular dynamics.

Technological improvements for signal integration are

continuously improving to follow reactions and transport within the complex matrix of the cell and its compartments. Electrochemistry

is

the

foremost

complementary

approach

for

single

cell

analysis.

Electrochemical conversion or a physical interaction of the analyte at an electrode surface generates the electrical signal (e.g. current, charge or potential).

The classic strategy developed by the

Wightman and Ewing groups positions a microelectrode near or at the surface of one cell for the electrochemical monitoring of individual exocytotic events.18,19 Here a background can be measured just prior to cell stimulation for reliable quantification of neurotransmitters during single and sequential exocytotic events. In this example, neuronal chemistry has been coupled to behavior in free-roaming animals. As compared with fluorescence imaging, this approach does not require a recognition probe to label the target which minimizes questions of cellular machinery perturbations. Moreover, since the electrochemical signal is mostly concentration-dependent, the electrochemical approach does not need achieve high spatial resolution at the expense of temporal resolution. Therefore, this method could provide a signal with both high spatial and temporal resolution. With these advantages, important information about the efflux of reactive oxygen/nitrogen species (ROS/RNS) have also been obtained at single cells contributing to the evolving elucidation of oxidative stress mechanisms as well as neuronal activity 20,21 Fast development in the ability to fabricate nanoelectrodes and the theory of nanoelectrochemistry has significantly improving detection limit, specificity, and resolutions of electrochemical measurement as reviewed in reference 22-25.

These achievements offer the feasibility of

investigating local events in single cells with high spatio-temporal resolution.

For better adaption in

biological and clinical study, a series of electrochemical microscopies with advanced resolution are emerging. Of particular note, nanoelectrode systems are expected to allow unprecedented analysis at the intracellular level. Moreover, detection at single living cell within biological systems such as the neocortex and retina has attracted attention because biological system links to single cell characteristics. In this prospective, electrochemical techniques developed in these frontiers including single-cell electrochemical imaging, intracellular analysis, and single-cell analysis within biological


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systems are summarized. Unique attributes of the methods and remaining technical challenges are discussed. Electrochemical microscopy for higher-resolution measurements Nanoelectrodes inherently exhibit high mass transport rates from radial diffusion and thus offer significant improvement in detection sensitivity for single cell analysis.26-28 The modification of novel sensing materials at the electrode surface is proposed to further improve sensitivity by facilitated electron transfer between the analyte and the nanoelectrodes.29-32

In particular, high temporal

resolution in the microsecond range is achieved so that fast neurotransmitter release from specific regions of a neuron, such as synapse, can be investigated. These achievements allow obtaining direct and important physiological evidences in local cellular communication. Given the importance of uneven cellular efflux associated with heterogeneous distribution of exocytotic microdomains in cells, high spatio-temporal resolution electrochemical mapping of exocytotic events or membrane molecules is exciting. Multi-site nanoelectrode arrays have been designed for high spatial resolution of exocytosis activity across single cells or clusters of single cells.33-36 The electrochemical images obtained from these arrays reveal subcellular temporal heterogeneities in exocytosis (i.e. cold spots vs hot spots) which was largely unprecidiented.37 For better adaption of electrochemical measurements in biological and clinic studies, electrochemical microscopy has become another trend for high spatio-temporal resolution analysis of single cells. Scanning electrochemical microscopy (SECM) is a classic electrochemical microscopy that uses a microelectrode/nanoelectrode in a precise tip position over a single cell and measures the current to obtain spatially resolved electrochemical signals.37-40 Due to small number of molecules being detected under nanoelectrodes, obtaining high-quality images of protein distribution or neurotransmitter release from regions of the single cell surface remains a challenge. Recently, a voltage-switching mode was adapted in SECM by switching the applied voltage to change the faradaic current from a hindered diffusion feedback signal (for distance control and topographical imaging) to the electrochemical flux measurement of interest (Figure 1A).41 Based on this switch, a steady-state current from a 6.5 nm nanoelectrode was collected in less than 20 ms so that membrane proteins on A431 cells and neurotransmitters from PC12 cells were visualized (Figure 1B). Moreover, to realize the simultaneous image of chemical and topographical information, fast-scan cyclic voltammetry (FSCV) was combined with alternating current scanning electrochemical microscopy (AC-SECM) to measure the impedance and faradaic current simultaneously.42

The

technique is demonstrated to image cellular respiration and topography without the addition of a redox mediator. Long-term topographical and chemical measurements during cell growth and degeneration


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are possible. Optimization in circuitry, noise reductions, and data processing will lead to further improvements in simultaneous multicomponent chemical and topographical imaging. Considering the challenging in the collection of very small current for SECM image, scanning ion conductive microscopy (SICM) has emerged over the past twenty years where the increase of access resistance between a nano-pipette in an electrolyte-containing aqueous medium and a poorly conducting surface is employed.43,44 Since this technique monitors the nanoampere ionic current flowing in and out of the micro/nano-pipette, it permits the determination of the surface topography at the nanometer-range without a highly-sensitive electrochemical instrument. The highest resolution of 3–6 nm was achieved by imaging S-layer proteins from Bacillus sphaericus on a mica surface with a 13-nm-diameter nanopipette.45 Currently, SICM has been wildly applied for biological investigations, such as morphological characterization, assessment of physiological activity, and dynamic observations of subcellular structures.46 As compared with the information collected from SECM, the absence of chemical information in SICM image suggests opportunities for continued innovation and further development of this technique to obtain chemical and topography information at a single cell level. To this end, a new SICM format was designed for simultaneous surface charge mapping and topographical imaging of cells (Figure 1C).47-49 In this strategy, the diffuse double layers at the nanopipette and the surface interact to create a perm-selective region leading to a polarity-dependent ion current. Surface-induced rectification is also performed as the bias is varied. As a result, the surface charge is elucidated by probing the properties of the diffuse double layer at the cellular interface.

The

combination

of

charge

and

topography

information

reveals

distinct

surface charge distributions across the surface of human adipocyte cells, whose role is the storage and regulation of lipids in mammalian systems (Figure 1D).48 The other strategy for introducing chemical information in an SICM image is the fabrication of a multifunctional dual-channel scanning probe nanopipette, which enables a simultaneous SICM and SECM image of the target molecules of living cells to be obtained.50-53 In the dual-channel probe, one barrel filling with electrolyte and the molecules of interest was open to the bulk solution for topographical feedback and local delivery to the cell. Meanwhile, a solid carbon electrode in the other barrel measured the local concentration and flux of the delivered molecules. Using a "hopping mode", the carbon electrode response was calibrated at each pixel in bulk for comparison to the measurement near the surface. This setup allows differentiation in the molecular uptake rate across several regions of single cells with individual measurements at nanoscale resolution.51


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Figure 1. (A) Schematic illustration of voltage-switching mode SECM; (B) Topography image of hippocampus neurons using constant-current mode SECM. Reprinted with permission from ref. 41. Copyright 2012 National Academy of Sciences. (C) Cartoons (not to scale) demonstrating the charge distribution around a negatively charged nanopipette in bulk (a), near a negatively charged surface (b), and near a positively charged surface (c) . (D) High-resolution surface charge map of a root hair tip with 50 nm pixel size. (a) An optical image of the scanned root hair with the scanned region represented by a black square; (b) normalized current at −0.4 V tip bias with respect to bulk. Reprinted with permission from ref. 48. Copyright 2016 American Chemical Society.

These two contemporary approaches, nanoelectrode array and scanning microscopy, provide high spatial resolution for single-cell analysis, but both have some technical restrictions. For example, the nanoelectrode array requires sophisticated fabrication protocols which are time-consuming. The small inter-electrode distance and thus possible chemical cross talk between adjacent electrodes limits the spatial resolution. For SECM or SICM, the use of a scanning nanoelectrode provides high spatial resolution at the expense of temporal resolution. Plasmonics-based electrochemical microscopy is a


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new tool developed in the past ten years that simultaneously allows wide-field imaging of the interfacial distribution of an electrochemical signal from a planar electrode with submicron spatial resolution and submillisecond temporal resolution.54,55

The principle of this electrochemical

microscopy is based on the sensitive dependence of surface plasmon resonance (SPR) on the surface charge density of a gold film (electrode), which changes with the applied voltage.

Since the

electrochemical impedance could be obtained from local surface charge associated with the modulating current, the plasmonic signals can be used for imaging the local electrochemical impedance.56 Using this SPR based electrochemical impedance microscopy (P-EIM) (Figure 2A), dynamic cellular processes, including apoptosis and electroporation of individual living cells, were studied. The simultaneously recorded bright-field optical and P-EIM images initially showed a rapid electroporation in the center of the cell, followed by a slower recovery process lasting for tens of seconds.57 In a more recent work, P-EIM was applied to study the endogenous G protein–coupled receptor (GPCR) stimulations of living HeLa cells with subcellular and millisecond spatial–temporal resolutions (Figure 2B).58 Despite the many unique features of this new technique, the expression of a P-EIM image contrast in terms of molecular-scale processes, particularly in cells, requires further research. The combination of P-EIM with fluorescence microscopy and other imaging techniques will help to develop a molecular-scale understanding of P-EIM imaging of cells. The parallel recording of signals from different regions of one cell allows the co-existence of high spatial and temporal resolutions; however, the inherent high detection limit associated with SPR complicates observation of low levels of chemical efflux or membrane molecules at singe cells. In our group, an electrochemiluminesence (ECL) microscopy technique was developed to image the membrane cholesterol or the efflux of hydrogen peroxide of single cells (Figure 2C).59-61 Electrochemiluminescence (ECL) is a highly sensitive electrochemical method using the emission light from the relaxation of electronically excited products to the ground state after an electrochemical reaction.62 In the presence of a luminescent probe, such as luminol, the molecule of interest in a single cell is converted by oxidase into hydrogen peroxide that generates luminescence. By the image of the luminescence from cells over the entire electrode surface, distribution information of molecules at the cellular membrane is obtained.63 The present setup can visualize the efflux of hydrogen peroxide from cells as low as 10 µM and is adaptable to most metal surfaces (Figure 2D). Since the relative long lifetime of oxygen-containing species generated from hydrogen peroxide during the luminescence process, cross-talk of luminescence from adjacent regions necessitates further electrochemical modulation to study the diffusion distance so that higher spatial resolution is realized.


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Figure 2. (A) Schematic illustration of P-EIM set-up; (B) the distribution of GlcNAc-containing glycoproteins was quantified by the SPR signal increment after WGA binding. Reprinted with permission from ref. 58. Copyright 2012 Nature Publishing Group. (C) Schematic illustration of ECL microscopy set-up; (D) images of Hela cells on an ITO electrode: (a) bright-field image; (b) luminescence image. Reprinted with permission from ref. 63. Copyright 2015 American Chemical Society.

Nanoelectrodes for intracellular electrochemical analysis The present single-cell electrochemical analysis primarily focuses on the detection of exocytosis activity in the cells or molecules at the plasma membrane. To promote the application of single-cell electrochemical analysis in biological studies, intracellular electrochemical analysis of single living cells needs to be resolved. This study requires the dimensions of the nanoelectrode to be as small as possible so that it can be inserted into the cell with minor interruption of cellular activity during electrochemical measurement.

However, in spite of higher current density generated at the

nanoelectrode, the dramatic decrease in the electrode size limits the electrochemical signal resulting in technical limitations for the intracellular application. The design of biocompatible nanoelectrode surface chemistries providing higher electron transfer rates is critical for improved utility.


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Remarkable progress was achieved in 2008 using a 42-nm polished Pt tip to penetrate immobilized cultured human breast cells for the initial intracellular voltammetry.64 The voltammetry observed at the nanoelectrode confirmed the characterization of intracellular redox states. Most importantly, the experimental result showed that an electrochemical probe with a tip radius approximately 1,000 times smaller than that of a cell could penetrate and travel inside a cell without apparent damage to the membrane. This key observation indicates the feasibility of intracellular electrochemical analysis using nanoelectrodes. In follow-up work, Pt black was electrodeposited inside an etched nanocavity to provide a sufficiently large electrochemical area, which yielded a stable and reproducible response to reactive oxygen species (ROS) and reactive nitrogen species (RNS).65 After positioning these nanoelectrodes in the cells (Figure 3A), weak and very short leaks of ROS/RNS from the vacuoles into the cytoplasm were detected (Figure 3B). The successful detection of biomolecules inside living cells promises advances in the investigation of oxygen stress processes. Moreover, by performing time-dependent quantitative amperometric measurements at different potentials, the relative concentrations of four key ROS/RNS in the cell cytoplasm and their dynamics were determined using this nanoelectrode.66 This achievement could be used to elucidate the chemical origins and production rates of ROS/RNS in cells at different states. To further improve the electrode material, a unique SiC-core-shell design to produce cylindrical nanowire electrodes was used for quantitative measurements of ROS/RNS in individual phagolysosomes of living macrophages.67 The electrode has superior mechanical toughness provided by the SiC nano-core and an excellent electrochemical performance provided by the ultrathin carbon shell. The successful modification of the nanowires at the tip of capillary might offer a new concept of nanometer-sized electrochemical sensors that couples the features of nanowires and nanoelectrodes for single cell analysis. For the quantification of vesicular transmitter content inside living cells, intracellular vesicle electrochemical cytometry was developed that included a nanotip conical carbon-fiber nanoelectrode to electrochemically measure individual nanoscale vesicles inside single neuron cells.68-70

The

approach involves the adsorption and subsequent rupture of vesicles on an electrode surface during which time the electroactive contents are quantitatively oxidized (or reduced).

Only part of

the vesicle content was released in typical exocytotic cases, which supports the intriguing hypothesis that the vesicle did not open completely during the normal exocytosis process, thus resulting in incomplete expulsion of its contents. Besides the classic disk nanoelectrode for intracellular electrochemical analysis, the nanocapillary has been used for this study.71,72 After pulling a quartz capillary to form a sharp nanopipette tip, carbon was pyrolytically deposited within the nanopipette shaft, generating a disk-shaped carbon nanoelectrode.73 The dimension of the nanoelectrode depended on the size of the nanopipette opening,


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which could be precisely tuned within 5–200 nm.

The continuous functionalization of the

nanoelectrode with platinum allowed electrochemical measurements inside the cell with minimal disruption to cell function. Most importantly, these nanocapillaries are adapted with an SICM setup, thus allowing high-resolution electrochemical mapping of species on or in living cells. Our group uses this nanocapillary to fabricate a nanokit for the assay on protein activity in single living cells.74 In our strategy, a nanometer-sized capillary was coated with a Pt layer and further insulated using wax. A thin layer of Pt at the tip not covered with wax was used as a ring electrode for electrochemical measurement of hydrogen peroxide. Meanwhile, the capillary was filled with the components from traditional kits, which was egressed outside the capillary by electrochemical pumping. After insertion of the capillary into the cells, femtoliter amounts of the kit components were

Figure 3. Monitoring ROS/RNS release induced by the mechanical stimulation of a macrophage. (A) Optical microscopic micrograph of a nanoelectrode inside a macrophage. (B) Typical amperometric current traces of the ROS/RNS release inside a macrophage induced by the insertion of a platinized nanoelectrode. Reprinted with permission from ref. 65. Copyright 2012 National Academy of Sciences. (C) Schematic of the nanokit used for the single-cell electrochemical analysis. (D) The faradaic charge of the electrode collected in J774 and HeLa cells. Reprinted with permission from ref. 74. Copyright 2016 National Academy of Sciences.


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egressed from the capillary and reacted with the analyte to generate hydrogen peroxide for the electrochemical measurement by the ring electrode (Figure 3C). Sphingomyelinase (SMase) activity in individual cells at different states was measured, and a high cellular heterogeneity of protein activity was observed (Figure 3D). The nanokit has adapted features of the well-established kits and integrates the kit components and detector in one nanometer-sized capillary, which provides a specific device for characterizing the reactivity and concentrations of cellular compounds in single cells. Single-cell analysis in a biological system Classical single-cell electrochemical analysis focuses on the development of new electrode architectures to achieve the measurement of single cultured cells. Although the exocytosis activity and molecule distribution of single cells have been extensively studied, research on the single-cell information responsible for animal behavior is seldom performed.75 Therefore, the introduction of the aforementioned electrodes and methods to measure a single cell in a biological system, primarily neocortex and retina, is significant in the fields of electroanalysis and electrophysiology. Currently, substrate-integrated microelectrode arrays are the main tool for the study of neuronal circuit-connectivity, physiology and pathology under in vitro or in vivo conditions.76 To fully decode the functions of individual circuit elements, simultaneous information on the identity, spatial location and wiring of neurons with great precision is required.

The achievement of simultaneous

electrophysiology and optical imaging could pave the way for high spatio-temporal resolution electrooptic mapping of dynamic neuronal activity. Accordingly, a graphene-based electrode array was fabricated to enable simultaneous optical imaging and electrophysiological recording (Figure 4A). High-frequency bursting activity and slow synaptic potentials were recorded that were difficult to resolve by multicellular calcium imaging (Figure 4B).77 Meanwhile, the present electrode position in the system faces some technical issues such as relative shear motion and chronic immune responses during long-term recording, which affects the spatiotemporal resolution during single-neuron measurement. To overcome these limitations, in vivo recording and a stimulation platform based on flexible mesh electronics were prepared and demonstrated stable multiplexed local field potentials and single-unit recordings in mouse brains for at least 8 months without probe repositioning.78 Although the use of a cell-non-invasive extracellular microelectrode array enables the simultaneous recording and stimulation of large populations of excitable cells for days and months78-80, this extracellular recording can only offer extracellular field potentials generated by action potentials. To deeply investigate the significant signaling between neurons that is mediated by subthreshold potentials (chemical or electrical synapses), intracellular recordings of synaptic potentials at single cells is important to achieve. A scalable intracellular electrode platform based on vertical nanowires has been established, allowing the intracellular recording and stimulation of neuronal activity in dissociated cultures of rat cortical neurons.81 In each nanowire, the silicon core and the metal tip


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provided electrical access to the cell’s interior, whereas the glass shell served as a material for tight sealing with the cell membrane. Due to its compatibility with silicon nanofabrication techniques, this platform provides a clear path towards simultaneous, high-fidelity interfacing with hundreds of individual neurons and mapping of multiple individual synaptic connections. Considering the serious fouling problem at the metal electrode during the analysis in the biological system, carbon nanotube (CNT) fiber electrodes were applied to record and stimulate the neurons in parkinsonian rodents. The result showed that these CNT electrodes stimulated neurons as effectively as metal electrodes with 10 times the surface area while eliciting a significantly reduced inflammatory response. Moreover, these electrodes can record neural activity for weeks and thus could be applied to the development of novel multifunctional and dynamic neural interfaces with long-term stability.82 Quantitative measurement is the other severe technical challenge for single-cell analysis in a biological system. Recently, a novel method to quantify octopamine release from the individual varicosities in the Drosophila larval system was developed.83 The detection takes advantage of the peripheral localization of the neuromuscular junction to facilitate the access of the microelectrode, and of the relatively superficial position of octopaminergic boutons within the larval body wall to enhance signal detection. Optical stimulation of type II boutons evoked exocytosis of octopamine, which was detected through oxidization at the electrode surface. Different types of release according to different shapes of the signals were observed, which might be related to the mechanism for opening the vesicle. Therefore, this system provides a strategy for quantifying the amount of transmitter released from single vesicles and for estimating the size of the vesicles in a biological system.

Figure 4. (A) Schematic illustration of a flexible graphene neural electrode array; (B) calcium transients (∆F/F0) for the electrode area (labelled ROI) increased to an elevated level (plateau) with respect to the baseline during the ictal-like events. Reprinted with permission from ref. 77. Copyright 2014 Nature Publishing Group.


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Conclusion This prospective aims at providing an overview of the current state of technology development with a focus of those methods that hold the most promise for future clinical driven studies.

While

great strides are being made in this realm, disconnects exist between developmental work on easily obtainable ideal systems and clinical work where actual disease states are being studied by those experts. In short, the tools being developed are being applied at an optimal pace and perhaps only collaboration between these fields can begin to maximize impact on the human condition through greater knowledge of life’s chemistry. Moreover, more cellular chemical and dynamics information at every specific location of the cell, including the easily accessible plasma membrane, cytosol, and its compartments is lacking in our current detailed characterization of cell biology and biochemistry. These challenging are pushing the development of non-invasive electrode architectures and electrochemical approaches to advance electrochemical imaging, intracellular analysis, and single within intact biological systems. Future work will clearly focus on further improving the spatiotemporal resolution of the methods. The use of novel materials and elegant design of recognition surfaces at the electrode should not be understated as a key aspect of needed work that is to be addressed concurrently with technical developments. The introduction of new physical principles and electrochemical/field modulation in electrochemical measurements will undoubtedly be a contributing factor over the next decades. Taken together, it is expected that these fabrication, chemical, and instrumental advances will spark efforts for even greater parallel-analysis of multiple analytes simultaneously. The establishment of multifunctional electrode surfaces and potential resolved approaches are also at for the forefront the work that allows a more comprehensive tool box for the biological scientist. Lastly, coupling of other analytical methods such as fluorescence, luminescence, and mass spectroscopy for tandem approaches will surely advance as well. It is our hope that this prospective will also contribute the establishment of these specialized technologies so that collaborations between analytical and clinical laboratories will advance knowledge of human disease states and eventually improve the human condition. Finally, these tools are expected to become available for routine use by clinical laboratories.

Acknowledgements We apologize in advance to all the investigators whose research could not be cited due to space limitations. We would like to acknowledge NSFC for support (nos. 21327902 and 21575060).


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