•REVIEWS• May 2020 Vol.63 No.5: 589–618SPECIAL ISSUE: Celebrating the 100th Anniversary of Chemical Sciences in Nanjing University https://doi.org/10.1007/s11426-020-9716-2

Single-entity electrochemistry at confined sensing interfacesYi-Lun Ying1†, Jiajun Wang1†, Anna Rose Leach2†, Ying Jiang4†, Rui Gao6†, Cong Xu3,5,Martin A. Edwards6, Andrew D. Pendergast7, Hang Ren8, Connor K. Terry Weatherly6,

Wei Wang1*, Paolo Actis2*, Lanqun Mao3,5*, Henry S. White6* & Yi-Tao Long1*

1State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University,Nanjing 210023, China;

2School of Electronic and Electrical Engineering, University of Leeds, Woodhouse Lane, LS2 9JT, Leeds, UK;3Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry,

the Chinese Academy of Sciences (CAS), Beijing 100190, China;4College of Chemistry, Beijing Normal University, Beijing 100875, China;

5University of Chinese Academy of Sciences, Beijing 100049, China;6Department of Chemistry, University of Utah, Salt Lake City, UT 84112, United States;

7Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States;8Department of Chemistry & Biochemistry, Miami University, Oxford, OH 45056, United States

Received January 9, 2020; accepted March 5, 2020; published online April 3, 2020

Measurements at the single-entity level provide more precise diagnosis and understanding of basic biological and chemicalprocesses. Recent advances in the chemical measurement provide a means for ultra-sensitive analysis. Confining the singleanalyte and electrons near the sensing interface can greatly enhance the sensitivity and selectivity. In this review, we summarizethe recent progress in single-entity electrochemistry of single molecules, single particles, single cells and even brain analysis.The benefits of confining these entities to a compatible size sensing interface are exemplified. Finally, the opportunities andchallenges of single entity electrochemistry are addressed.

single entity, electrochemistry, confining effect, sensors, nanopores, nanoparticles, single cell, brain

Citation: Ying YL, Wang J, Leach AR, Jiang Y, Gao R, Xu C, Edwards MA, Pendergast AD, Ren H, Weatherly CKT, Wang W, Actis P, Mao L, White HS, LongYT. Single-entity electrochemistry at confined sensing interfaces. Sci China Chem, 2020, 63: 589–618, https://doi.org/10.1007/s11426-020-9716-2

1 Introduction

Why do we need to develop single entity electrochemicalmeasurements? Because the precision and depth of our un-derstanding of chemical systems are determined by analy-tical science. In traditional ensemble-based studies (cyclicvoltammetry, and coulometry), one often measures theaveraged activity of an interface containing millions and

even billions of elementary entities (e.g., molecules, nano-particles or cells). These ensemble measurements are sub-sequently analyzed to extract some averaged values, todescribe one or several chemical features. For instance, themorphology and size of nanoparticles and cells are typicallyevaluated by electron microscopes. Several energy spectro-scopies and elementary analysis techniques are available todetermine the chemical composition of the nanoparticle/cell.Although powerful, these ensemble-based approaches relyon averaged activities and averaged structural/conformationfeatures, which dilute the intrinsic heterogeneity and blurstructure-activity relationships (SAR) [1-4]. Therefore, ad-

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†These authors contributed equally to this work.*Corresponding authors (email: wei.wang@nju.edu.cn; Actis@leeds.ac.uk;lqmao@iccas.ac.cn; white@chem.utah.edu; yitaolong@nju.edu.cn)

vancements in understanding elemental chemical processesand complex chemical reactions are significantly hinderedby examining ensembles of entities.The characterization of individual molecules in bulk so-

lution could help identify rare subpopulations, hidden in-termediates, dynamic interactions and multiple reactionpathways [5]. Monitoring single biomolecules deepens theknowledge of fundamental chemical reaction processes andfacilitates precise diagnosis of early-stage diseases [1, 3, 6,7]. The evaluation of electroactive nanomaterials at singleparticle level could help to explore the intrinsic hetero-geneity of the nanomaterials in terms of both structure andactivity [2, 8, 9]. Since the size, morphology, chemicalcomposition, facet, crystallinity, defect and surface chem-istry are dramatically different from one nanoparticle toanother, the electrochemical activities of individual entitiescould vary by orders of magnitudes [10]. Single nanoparticlemeasurements have found numerous applications in broadfields such as batteries, fuel cells and sensors [7, 11, 12]. Theinvestigation of a single cell allows one to explore in-tracellular and intercellular biochemical process and to un-cover the fundamental differences that exist between cells[13]. It provides techniques that could be used for betterunderstanding cellular networks and even tissue function.Regarding the measurement of single tissues, the brain is themost sophisticated organ to be explored. Determining themolecular processes that underpin the development, func-tion, and pathology of the nervous system, however, can bevery challenging [14].How can one clearly “see” entities, one-by-one, in the

bulk? Electrochemistry is a sensitive methodology whichconverts the characteristics of single entities into the rapidelectrochemical response (Figure 1). The electrochemicalsignature signals refer directly to the single analyte proper-ties such as redox properties, structure, charges and sizes.Compared to the conventional optical microscopy, for in-stance, which has a detect limitation of ca. 200 nm [15], theelectrochemical approach with nanoscale sensing interfacecould feasibly achieve nanometer scaled spatial resolution[2]. Moreover, energy spectroscopy usually requires ultra-high vacuum conditions, but provides relatively low tem-poral resolution [16]. However, the development of elec-trochemical instrumentation has provided us with ultra-hightemporal resolution with high current sensitivity, approach-ing femto-ampere levels [17]. These electrochemical signalscontain rich dynamic information from single entities, whichcould help to understand the fundamental unit of a chemicalsystem.The main challenges in the electrochemical measurement

of single entities is the construction of an appropriate sensinginterface [18, 19]. Traditionally, macroscale interfaces reportthe ensemble features of target analytes. If a micro/nano areais taken from the traditional macroscale sensor, it could then

turn into a confined structure for reducing the size of thesensing interface [20]. Then, the size of the sensing interfacecould be designed to be compatible with sensing a singletarget. The suitable sensing interface could be a single par-ticle, a planar nanosized electrode or a nanochannel. Forexample, nanometer-sized pore-forming biomolecules (na-nopores) can provide a well-defined space for accom-modating of a single molecule [20, 21]. As a single moleculeenters a nanopore, it modulates the ionic flux though thepore, resulting in an ionic fluctuation with sub-millisecondtemporal resolution and near atomic spatial resolution [5, 6,22-29]. For detecting single nanoparticles, nanometer sizedelectrodes and nanopipettes have been fabricated, which al-low detection of one single nanoparticle at a time on itssurface [7, 10-12, 15, 30, 31]. Moreover, nanometer sizedtips provide high spatial resolution for extracting the contentof individual cells as well as for high-resolution imaging oflive cells. The micro-sized and chemically engineered elec-trodes show remarkable advantages in electrochemical de-termination of neurochemicals in the brain by overcomingthe challenges of in vivo selectivity and stability. With anappropriately designed sensing interface, electrochemicalapproaches offer one of the most sensitive and effectivetechniques that can be employed to acquire new knowledgeof chemical systems.The term “single-entity electrochemistry” was first coined

in 2016 at the Faraday Discussion at York, UK, whichbrought together scientists from different branches of elec-trochemistry to discuss diverse approaches and techniquestoward similar goals [1]. As mentioned, the single entityinterrogated by sensitive electrochemistry requires an ade-quate sensing interface [29, 32]. For single molecule/nano-particle analysis, the nano-interface is a necessity. In 2018,the Faraday Discussions themed “Electrochemistry at nano-interface” was held at Bath, UK. There, it was noted that thedevelopment of a well-defined nano-scale sensing interfaceis still a challenge for single-entity electrochemistry. In thisreview, we summarize the recent progresses and goals ofsingle-entity electrochemistry for analyte scales as small assingle molecule, nanoparticle, cell and even single tissue(Figure 1). Then, we discuss the applications for single-entityelectrochemistry to solve the questions from the biologicaland chemical processes, including measurements performedin the brain, and highlight electrochemical methods whereprecise descriptions of complex electrochemical process aredetermined with high temporal and spatial resolution. Fi-nally, unmet challenges and future opportunities for the fieldof single-entity electrochemistry are discussed.

2 Nanopore-based single molecule analysis

Single molecule sensing requires a compatible sensing in-

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terface to provide sufficient amperometric resolution andtemporal resolution. Confining a single analyte into a sui-table space, assembled by sensing elements and without in-terference, from neighbor analytes, is the ideal case for singlemolecule sensing [5, 29, 33]. A nano-scale cavity, nanopore,is an ideal model of such space. By applying a bias voltage,the single analyte together with conducting ions are confinedinto such an interface that is spatially suitable to singlemolecule allowing label-free detection. To establish such ananopore, the membrane channels are chosen and withcareful self-assemble process. The whole sensing interface ofbiological nanopore is composed by one single membraneprotein, which could be regarded as single-biomolecule in-terface (Figure 2(a)). The volume of this single biomoleculeinterface is comparable to the single target molecule.Therefore, a single biomolecule interface captures andidentifies one-single-molecule-at-a-time. By tunning theevery single single amino acid residue inside single-biomo-lecule interface at atomic level using site-direct mutation, theresulted interaction to the single analyte could be manipu-lated towards better sensitivity and selectivity. Specifically,as illustrated in Figure 2(b-d), the cap domain of the Aero-lysin is critical to selectively capture the single moleculewhilst the β-barrel region is responsible for sensitively sen-sing the target single molecules. The proof-of-principle ex-periment was performed by using α-Hemolysin, a toxinsecreted from S. aureus, probing polynucleotides in 1996[34]. Later on, the solid-state materials (e.g. SiNx [35-37],graphene [38-40], MoS2 [41]) have been used to mimicbiological membrane channels. The solid-state nanoporeowns unique favorable characteristics such as facile surfacemodification, controllabe diameters and the capability ofintegrating with other electrical or optical detection tech-niqes [42]. The reader could refer to reviews in refs [43-45]for detials regarding the fabrication and application of solid-state nanopores.Biosensing in the single biomolecule interface for single

molecule detection has been undergoing for almost threedecades. The first nanopore workshop themed ‘Biosensingwith channel’ workshop was held in Bremen, Germany in2006. Faraday Discussion collected the research interestthemed “Electrochemistry at a nano-interface” [18], typi-cally with a section of ‘Processes at nanopores and Bionano-interface’. Aims to establish emerging electrochemicaltechniques for characterizing dynamic processes at func-tional nanopores. This is possibly been integrated with noveloptical techniques and the development of ultrafast currentmeasurements. With the intensive interaction made by theseinternational nanopore conference, we expect that the newknowledge acquired by nanopore technology will providenew inspirations and directions for single-entity electro-chemistry.

2.1 Construction of the single biological interface (ex-ploring and sensing optimization)

Usually, the nanopore confined space is composed by singlebiomolecules such as α-Hemolysin [46], Aerolysin [47],MspA [48], OmpG [49], CsgG [50], Lysenin [51], FhuA[52], YaxAB [53], phi 29 [54], and BPV E1 helicase nano-pore [55], with some of the examples shown in Figure 3.Biological nanopores are natively functionalized in the

cellular membrane. In order to take the usage of these bio-molecules for the purpose of single molecule probing, anartificial phosphor lipid bilayer is established. Phosphor lipidusually dissolved in the alkane, typically decane for Muellerand Rudin Method. Otherwise, the Montel and MuellerMethod [56] is used to form solvent-free bilayer. The firstapproach contains solvent that is more flexible to membraneprotein with different thickness of hydrophobic domain,while the latter approach requires a standard pretreatment tothe bilayer support, which provides the advantage to buildasymmetric bilayer. Both approaches can form a free-standing lipid bilayer that sits on an aperture with diameter of

Figure 1 Road map of single-entity electrochemistry approaches. The nanopore interface is a water filled pore that allows ions (cation (P+) and anion (N-))bypass. The pore lumen fits the nanometer scale analytes and probes their characteristics. The nucleation and growth of a single nanobubble analyze with ananoelectrode. The single nanoparticles (NP) are probed by the nano/micro-scale electrode. Single cell is clamped by nanopipette-based electrochemicalmeasurements. Electrochemical reaction at the cellular surface or intracellular biomolecules is characterized via the electric responses. In vivo analysis of thetissue is performed by the electrode array. By positioning the array at the region of interest, the signal allows to characterize the electrophysiologicalbehaviour (color online).

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50-100 μm, which is composed from material such as PTFE.The free-standing lipid membrane is also called ‘black lipidmembrane’ (BLM) named as such because the membranedoes not reflect light during the thinning out process [56].The self assembly of nanopores from biological material is

more sophisticated. Taking Aerolysin nanopore as an ex-ample, the aqueous soluble biomolecule undergoes hepta-merization, post-prepore, then extends its inner barrel toform a pore with water-filled lumen [47]. During the ex-perimental process, the oligomeric Aerolysin biomolecule isadded directly to the BLM forming nanopore with the aid ofa membrane bias voltage at negative polarity [57]. The so-phisticated membrane protein purification and reconstitutioncan also be overcome by using native bacterial outer mem-brane vesicles fusion with artificial lipid bilayer [58].The biological nanopore is adjustable in pore diameter

with high reproducibility for analytes with difference sizes.For example, the introduction of the β-strands increases inpore radii from 1.6 nm for FhuA [52] Δ1-160 up to a max-

imum of about 2.7 nm for 8 β-sheet insertions. In addition,WT FhuA and its mutants W116S and W112SW116S aremutagenized and obtained the nanopore opening with dia-meter 1.6 nm, 1.1 nm, 0.84 nm, respectively. Aside from theconfined effect provided by a nanopore that fits to the analyteat single molecule level, the probing interface can also beprecisely manipulated by using the design concept of “sin-gle-biomolecule interface”. (Figure 2(b-d)). Figure 4 listedso far engineered Aerolysin, by manipulating the amino acidat the sensing interface, where the weak interaction betweenthe probes and analytes is fine tuned for better sensitivity andselectivity [59-61].

2.2 Nanopore based single-molecule sensing applica-tions

2.2.1 Nanopore for DNA sequencingSince the completion of human genomic protein in 2003(https://web.ornl.gov/sci/techresources/Human_Genome/

Figure 2 (a) The transformation of a sensing interface into a single molecule interface. (i) The macroscale interface with a sensing molecule for detectionof the targeting analyte. (ii) The macroscale interface shrinks and turns over, (iii) then rolls into a channel as the micro-/nanoscale interface. (iv) A single-biomolecule interface is derived from a single recognition molecule at the nano interface. A self-assembled aerolysin membrane protein is regarded as anexample of a single-biomolecule interface. The oligonucleotide is taken as an example for illustrating the analyte. (b) Example of analysing an oligonu-cleotide through a single aerolysin interface with site-direct mutagenesis. A wild-type aerolysin with two positively charged amino acids produces distin-guishable blockage current (red box). The speed of a single negatively charged oligonucleotide translocation aerolysin pore is as slow as ∼ 2 ms/base due tothe strong electrostatic interactions caused by the positively charged residues at the entrances and inside the lumen. (c) The mutant I with negatively chargedglutamic acid located at the entrance of the pore, which generates a high entropic energy barrier for the sensing of negatively charged oligonucleotidemolecules. Consequentially, barely no current blockages occur with the continuous time-current recording. (d) The mutant II with negatively chargedglutamic acid at the lumen of the single-molecule interface leads to a prolonged duration (red box). The dynamic conformational changes of the oligonu-cleotide may be enhanced inside the pore due to the electrostatic repulsion, inducing further current oscillations (blue box). Reproduced with permission from[20] (color online).

Figure 3 Side and top views of nanopore self-assembled from biological components and the corresponding pore diameter. Narrowest pore opening isindicated in red. The membrane domains where the proteins assemble are hinted by solid lines (color online).

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project/index.shtml), DNA sequencing technique are devel-oped in the direction of maximized base per read, cost effi-ciency and time saving. More importantly, such tool can beused to understand increasingly complexity of phenotypes[62]. The commercialized DNA sequencing tool based onnanopore has shown its great potential [63]. CsgG is engagedwith the next generation of ssDNA sequencing [64] and in2016, the nanopore based DNA sequencer could reach 450bases per second and enabling 10Gb DNA sequencing datato be obtained. At the current state, further progress is es-tablished in length discrimination of oligonucleotide [74](Figure 5(a)), dsDNA detection [65, 66] (Figure 5 (b)) aswell as RNA sequencing [67-69] (Figure 5 (c)).

2.2.2 Nanopore beyond DNA sequencingBeyond the sequencing, nanopore based single molecule toolcould be used to analyze small molecules, peptides, lightdriven reaction, chemical reaction, protein folding/unfoldingprocess, enzymology, binding process etc. (Figure 5 (e-g)) asdiscussed below.Small molecules are difficult to be distinguished with the

conventional approaches due to the demand of sub-mole-cular resolution. The nanopore, towards this hinderance,provides compatible confined space for sensitive electro-chemical measurement, resulting molecular resolution andhyper sensitivity. The α-Hemolysin could detect melaminewhich is a common food additive as low as 10 pM with theaid of DNA probes [70]. Under de novo design by per-forming mutagenesis, label-free detection is achievable. Inthe case of monosaccharides, D-glucose and D-fructose existin aqueous solution as an equilibrium mixture. By control-ling the engineered point inside the a-Hemolysin nanopore, aboronic acid reacts reversibly with D-glucose as the pyr-

anose isomer (α-d-glucopyranose) and d-fructose as eitherthe furanose (β-d-fructofuranose) or the pyranose (β-d-fructopyranose). The outcome allows one to discriminate theindividual sugar at single molecule level [71]. On the otherside, increasing the interaction between the small moleculesand sensing interface could also enhance the discriminateresolution. The K238Q Aerolysin successfully discriminatethe cysteine and homocysteine due to the strong interactionprovided by the substitution by Glutamine (Q) [72]. Azo-benzene is a UV-sensitive molecule, by modifying it to theoligonucleotide (Azo-ODN), the cis-to-trans or trans-to-cismodification under the UV-Vis control could be real-timemonitored by Aerolysin composed confined space [73] ((Figure 5(e)).Peptides are usually chosen as the biomarkers to diseases.

Unlike the DNA segments which are usually negativelycharged, the charge density of peptides becomes an im-portant descriptor as well as the 20 amino acid components.The first peptide sequencing by nanopore based electro-chemical approach is performed with α-Hemolysin andAerolysin [79, 80]. This work has demonstrated the potentialof pore-forming material to detect the peptides and guide theway to design such material for better sensitivity and se-lectivity. With a prominent example of nanopore sensingdisease related peptides, neurodegenerative disorder dis-eases-related peptides are analyzed by the nanopore labelfree technique. Alzheimer’s disease, Parkinson’s disease andthe others are associated with the presence and activity oftoxic protein aggregates known as amyloids. Common stra-tegies to analyze the biophysical properties of these abnor-mal aggregations are Ion mobility spectroscopy-massspectroscopy (IMS-MS), Fluorescence correlation spectro-scopy (FSC), cryo-transmission electro-microscopy, Atomicforce microscopy and NMR etc., however these techniquesprovide ensemble information. Nanopore utilized electro-chemical signal to analyze the toxic protein aggregates in-asmuch the single molecule resolution and label free merits(Figure 5(d)) [27, 81, 82]. Specifically, β-amyloid (Aβ)peptides (Aβ25–35 and Aβ35–25) were distinguished in real-time due to their characteristic blockades. The understandingof the nanopore probing metal-induced folding of peptides orother natural folding states of DNA or peptides at closephysiology condition were demonstrated [27, 83]. For in-stance, the Aβ peptide conformational changes induced bymetal binding (Cu2+ and Zn2+) are studied and quantified thebinding affinity [84]. Other peptide segments owe similarmolecular weight but opposite net charge could be easilydistinguished, thanks to the nanopore confined space whichenhances the electrostatic interaction between analytes andsensing interface [85]. Peptides with single amino acid dif-ference in length could also be sensitively discriminated byWT Aerolysin nanopore [86] and FraC nanopore [87].The chemical reaction process (e.g. cysteine chemistry,

Figure 4 Manipulation of probes at the nanopore based single-biomole-cule interface: Possible sites at hot sensing regions of Aerolysin have beenmutagenized [59-61] (color online).

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phosphorylation etc.) could also be investigated at suchbiological interface (Figure 5(f)). For example, the disulfateconjugation between analytes and engineered nanoporeshows the potential to directional control the moleculemovement protensively [77, 88]. The reaction between cy-steine and thiol-directed PEG molecule inside the OmpFnanopore could be monitored during the experiment [88].Rather than the characteristic cysteine chemistry, the click

reaction between azide and alkyne at the presence of Cu2+

inside a nanopore could also be real-time monitored withlabel-free merit [89]. The reversible dynamic covalent imi-noboronate formation inside the nanopore towards the re-active behavior of the lysine residue located at the poreopening and constriction was studied individually [90].During the oligonucleotide moving through the nanopore

lumen, single phosphorylation at different positions can be

Figure 5 Examples of nanopore based single-entity measurements. (a) ssDNA [74]; (b) dsDNA [75]; (c) RNA [34]; (d) Peptide [76]; (e) Light drivenmolecular structure modification [73]; (f) Chemical reaction [77]; (g) Enzyme cleavage monitoring [78] (color online).

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distinguished using Aerolysin by reading out the currentstepwise fluctuation [91]. Moreover, distinguish currentsignature from α-Hemolysin could even tell the varientsbetween unphosphorylated, monophosphorylated and di-phosphorylated protein (TrxS112−P-oligo(dC)30) [92]. Thesensing capability could be optimized by utilizing other na-nopore varieties such as CsgG, MspA, Aerolysin, FhuA etc.for varies application.The concept of nanopore based enzymology is addressed

[93, 94]. Basically, the enzyme relation biochemical processcan be monitored by nanopore based single entity chemistrywith provides label-free merits at single molecule resolution(Figure 5(g)). Trypsin is a serine protease which cleavespeptide chains at carboxyl side of the lysine and arginine.The process of Aβ cleavage by trypsin is real time monitoredusing α-Hemolysin by reading the step-wise signature cur-rent without labeling [95]. As another case, Exonuclease I isa DNA specific enzyme which catalyzes the removal ofnucleotide from linear ssDNA in the 3’ to 5’ direction. TheAerolysin can monitor the stepwise cleavage of oligonu-cleotides by Exonuclease I (Figure 5 (a)) [74].Pore-forming material is not meant to sense every typical

molecule, and the probe interface requires de novo design.Taking Aerolysin as an example, based on the structuralmodel of such nanopores, the sensing domain for ssDNAtranslocation has been mapped and demonstrated that R220and K238 are the critical sensing regions [96]. A series ofmutants K238C, K238Y, K238F and K238G are then per-formed and demonstrated that dynamic interaction betweenanalytes and pore interface enhances the better sensitivity forsingle-molecule analysis (Figure 4) [60]. Based on previousresults, the weak interaction between the critical sensingregion and the nucleotide is vital to the base sensitivity; as aresult, the Glutamine is chosen due to the strong interactionwith nucleotide bases. As expected, the single lesion nu-cleotide base in a mixture of heteronucleotide could be dis-criminated by K238Q [97]. As another case, the introducing

of cysteine in the Aerolysin, K238C, is engineered tomonitor the formation of disulphide bond with 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) with stepwise resolution ow-ing to formation of disulfate bonds [98]. Furthermore, theprecise introduction of cysteine into the α-hemolysin nano-pore allows monitoring the individual 0.7 nm steps of asingle molecular hopper under the control of electric field[77]. The mutant MspA, M2-NNN MspA, with the aid ofphi29 polymerase (DNAP) could control the passage ofDNA strand through the nanopore thus boosting the directdistinguish of nucleotide bases [99]. Such mutation alongwith substrate specified polymerase provides an approach toDNA sequencing. Not only the DNA sequencing, pointmutagenesis at E181C of OmpF along with cysteine chem-istry allows directional identification of small moleculesthrough the nanopore, which is a conundrum for drugscreening in nanopores [100].

3 Single nanoparticle electrochemistry

The challenge here is to resolve the tiny reaction currentfrom single electroactive nanoparticles from the contribu-tions from other individuals as well as the background cur-rent (for instance, charging current and polarization current).Because the electrode current itself is blind and has no spatialresolution at all, one has to ensure that there is only oneeffective nanoparticle at the electrode surface at the momentof measurement, and to minimize the background current byreducing the size of electrode. Three types of strategies havebeen proposed to reach this goal as illustrated in Figure 6. Inthe first strategy (Figure 6 (a)), a nanoelectrode was fabri-cated with an extremely small effective size down to tens ofnanometers. A single electroactive nanoparticle with com-parable diameter was formed on the nanoelectrode via post-immobilization or in situ electrodeposition. Because therewas only one nanoparticle and the background current was

Figure 6 Schematic illustration of three types of strategies for studying single nanoparticle electrochemistry (color online).

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almost completely suppressed, any electrode current shouldbe attributed to the nanoparticle on the electrode. In thesecond strategy (Figure 6 (b)), a microelectrode was held atan appropriate constant potential to reach a stable and smallbackground current. Very diluted nanoparticle suspensionwas used to allow for discrete and stochastic collisions ofsingle nanoparticles onto the microelectrode surface, leadingto a transient disturbance to the baseline current. The cor-responding spike in the current profile was able to reveal theactivity of the single nanoparticle in the collision event. Thekey point of collision strategy is to temporally separate dif-ferent nanoparticles according to the different times of col-lision. In the third strategy Figure 6 (c), a movablenanoelectrode scanned over a substrate surface on whichmany single nanoparticles had been previously immobilizedwith a very low number density (coverage). The nanoelec-trode-substrate distance was so close that only the individualnanoparticle covered by the nanoelectrode was able to ef-fectively transfer electrons. Consequently, the recordedcurrent can be assigned to a particular individual accordingto the spatial location of the nanoelectrode. Since this fieldhas been increasing so rapidly, interested readers are referredto these valuable review articles for a comprehensive un-derstanding [7, 10-12, 15, 31]. Here, we provide frameworkdescriptions with particular emphasis on the milestones andthose most recent studies.

3.1 Nanofabrication

The most straightforward solution to study single nano-particle electrochemistry is to fabricate a nanoelectrodewhich contains only one nanoparticle on its surface. In orderto minimize the background current, it is critical to make surethat the size of nanoelectrode is smaller than, or at leastcomparable with, that of the nanoparticle. In one of theearliest attempts, Zhang et al. immobilized a single 15-nmgold nanoparticle onto a 10-nm platinum nanoelectrode byvirtue of electrostatic adsorption [101]. The successful fab-rication was evidenced by transmission electron microscopycharacterization, which also revealed the actual size of thenanoparticle (Figure 6(a)). By using oxygen reduction re-action as an example, size-dependent catalytic activity wasinvestigated at single nanoparticle level [101, 102].Engineering the nanoparticle-electrode interface plays an

essential role here. It must allow for efficient electrontransfer between surface-attached nanoparticles and nanoe-lectrode, and at the same time reach a near-unity surfacecoverage to suppress the background current. Schuhmann etal. recently developed an in situ method to synthesize a co-balt-based single metal-organic framework (MOF) nano-particle on carbon nanoelectrode [103]. The nanoparticleunderwent further thermal pyrolysis to produce cobalt-basedactive sites for oxygen evolution reactions in alkaline solu-

tions. The introduction of single MOF nanoparticle not onlyprovided a well-defined geometry during catalyst prepara-tion, but also helped the characterizations on the amount anddistribution of Co-based catalytic sites.Because of the heterogeneity, it would be difficult to build

a solid SAR by studying only one or a few nanoparticles.Instead, statistical analysis to a sample set containing thou-sands and even more individuals is usually beneficial to re-veal a reliable SAR. In order to achieve this goal,nanofabrication strategy still needs to further improve itsefficiency and throughput.

3.2 Single nanoparticle collision

The collision of a single nanoparticle at an ultra-microelectrode disturbs the electrode current. By analyzingsuch disturbance, the activity of this particular nanoparticlecan be evaluated. According to different principles, threetypes of collision events have been well studied so far, in-cluding the blockade mechanism for electrochemically inertnanomaterials, the direct electrochemistry mechanism forredox active nanomaterials, and the catalytic amplificationmechanism for electrocatalytic nanomaterials.In the blockade mechanism, the presence of an electro-

chemically inert nanoparticle not only reduced the effectivesurface area of electrode, but also altered the mass transportcondition, resulting in the gradual decrease in the current[104]. The current profile carried rich information regardingthe property of nanoparticles as well as its interaction withthe electrode. For example, if there was a strong attractiveforce between the nanoparticle and the electrode, a hit-and-stay type collision was expected, accompanying with astaircase decrease in the current profile. In the oppositescenario, no collision can be observed at all if there was astrong repulsion. Under appropriate conditions, hit-and-runtype collision can be observed as evidenced by the spike-likenegative peaks in the current profile. The residence time(peak width) was an important parameter to explore the na-noparticle-electrode interaction. In addition, statistical ana-lysis on the collision frequency and intensity (peak height orarea) has proven powerful to calculate the molar con-centration and size of nanomaterials, respectively. Earlystudies often focused on latex nanobeads and nanodroplets.Increasing attention has been recently paid to biologicallysignificant nanoparticles such as microbials [105] and viru-ses [106].In catalytic amplification mechanism, the collision of an

electrocatalytic nanoparticle formed a transient catalytic siteto increase the electrode current in the presence of reactants[107]. In addition to the popular metal-based electrocatalyst,an interesting new trend is the photoelectrochemical detec-tion of semiconductor nanoparticles [108, 109]. Under theillumination of light, collision of semiconductor nano-

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particles facilitated the charge-carrier separation and in-creased the photoelectrochemical current in the presence ofsuitable scavengers.The colliding nanoparticle itself could undergo electro-

chemical oxidation or reduction to contribute to the electrodecurrent directly. A typical example was the oxidative strip-ping of metal nanoparticles such as silver and copper [110].Because of the extraordinary reactivity of silver nano-particles, it served as a very popular choice to study theinterfacial electron transfer as well as the nanoparticle-electrode interactions. An inversed approach was recentlyproposed to achieve the reductive synthesis of metal nano-particles upon the collision of single micelles containingmetal salt precursors [111]. In addition to metal nano-particles, the impact of Li-ion storage metal oxide nano-particles onto the electrode was also investigated driven bytheir practical significance in Li-ion batteries [112]. Thisprocess involved the intercalation of Li+ into the nano-particle. As a modified form of direct electrochemistry, ve-sicles [113] and liposomes [114] containing redox moleculesinside the lipid bilayer membrane could release its cargoupon collision, resulting in the increase in the reaction cur-rent.There are two standing challenges for single nanoparticle

collision in SAR studies. First, while the activity of singlenanoparticles can be determined from the discrete peaks inthe current profile, the structural feature associated with theparticular peak is completely missing. Combination of op-tical microscopes could be a useful development to recordthe location of collision for further structural characteriza-tions [101]. Second, a constant potential was often applied tothe electrode to suppress the charging current, so that thisstrategy was not quite compatible with the potential sweep-ing techniques.

3.3 Scanning electrochemical imaging

Scanning electrochemical microscope (SECM) has demon-strated its capability to study single nanoparticle electro-chemistry by approaching a nanoelectrode towards asubstrate on which nanoparticles were sparsely immobilized[104]. A very promising derivation of SECM is scanningelectrochemical cell microscope (SECCM). In this config-uration, counter and reference electrodes were placed in apipette with a tip size of tens of nanometers. When thepipette was filled with solutions and approached to thesubstrate, a droplet was formed with a contact radius at mi-crometer scale, which defined the size of effective electrode.When only one surface-immobilized nanoparticle was cov-ered by the droplet, the recorded electrode current could beassigned to this particular nanoparticle. When comparingwith nanofabrication approaches, SECCM exhibited sig-nificantly improved throughput as the nanopipette can freely

hop over the substrate. In a recent report, Unwin and cow-orkers [115] have shown that the cyclic voltammetry ofsingle LiMn2O4 nanoparticle can be reliably determined. Byvirtue of the programmed scanning of nanopipette, the re-activity of hundreds of nanoparticles can be recorded in aone-by-one manner.

3.4 Optical electrochemical imaging

In addition to these current-based approaches, optical elec-trochemical imaging represents an alternative and com-plementary approach to indirectly but quantitatively studysingle nanoparticle electrochemistry by coupling opticalimaging techniques. The basic methodology of opticalelectrochemical imaging was thoroughly summarized in arecent review article [15]. The brief concept was illustratedin Figure 7. In this configuration, nanoparticles were spar-sely immobilized on an optically transparent electrode, suchas ITO and ultrathin gold film, which was placed on aninverted optical microscope. With the development of opticalimaging techniques, there have been various kinds of opticalmicroscopes that can image single nanoparticles according tothe corresponding optical and spectroscopic principles. Op-tical images of these nanoparticles were continuously re-corded to produce a series of time-lapsed images. The opticalintensity of each and every nanoparticle was monitored inparallel to report its potential-dependent optical or spectro-scopic property. For example, a typical cyclic voltammetryscan to the electrode would lead to the transition betweenoxidized and reduced states of the nanoparticle, which wasindicated by the reversible alternation and recovery of itsoptical intensity. A suitable optical to electrochemical con-version model was required to quantitatively extract theelectron transfer rate, i.e., the current, of single nanoparticlesfrom its optical curve. In order to distinguish from the di-rectly recorded electrode current, one often uses the term,optical current, to describe the indirectly calculated results.Here the potentiostat was solely used to apply the potentialand to enable the reactions. The reaction current of singlenanoparticles was completely calculated from the opticalsignals. Recent studies have demonstrated the validity ofsuch conversion by comparing the simultaneously recordedelectrochemical current and optical current [112, 116, 117].The merit of optical electrochemical imaging relied on the

rational design of optical to electrochemical conversionmodels. As one of the most basic type of optical imagingtechniques, bright-field transmission microscope measuresthe extinction of single nanoparticles. It is therefore sensitiveto the absorption spectrum (color). By monitoring the elec-trochromic kinetics of single tungsten oxide nanorods (apromising material used in smart windows) under bright-field transmission microscope, Sambur and coworkers dis-covered that a particle-dependent waiting time was required

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prior to the color change [118]. Dark-field microscope is apowerful format to visualize single plasmonic nanoparticlesbased on its scattering. It has been demonstrated that thescattering intensity of silver nanoparticles was a function ofits volume, and thus the number of silver atoms. Accordingto the dependence, one was able to resolve the reaction rateof single nanoparticles during electrochemically assisteddeposition or oxidative stripping [117, 119-121]. Singlemolecule fluorescence microscopy [122], Raman micro-scopy [123] and electrochemiluminescence microscopy[124] were also adopted to evaluate the electrochemical ac-tivity of single nanoparticles by detecting the fluorescence,Raman and electrochemiluminescence signals of the productmolecules, respectively. Surface plasmon resonance micro-scopy is a wide-field optical microscopy that is sensitive tothe refractive index nearby an ultrathin semi-transparent goldfilm. The electrocatalytic reduction of protons at a platinumnanoparticle formed hydrogen molecules, which decreasedthe local refractive index. Tao and coworkers [125] deducteda quantitative relationship to calculate the catalytic current ofsingle nanoparticles out of the optical intensity curves. Wanget al. [126] recently discovered the dependence of the re-fractive index of single LiCoO2 nanoparticles on its lithiationstate during electrochemical cycling, allowing for measuringthe optical cyclic voltammogram of single Li-ion storagenanoparticles.In addition to these Faradaic reactions, non-Faradaic

charging of single metal nanoparticles and graphene can alsobe investigated because the dielectric constants of thesematerials were dependent on the electron density. The lattercan be precisely regulated during electrochemical chargingand discharging. Mulvaney et al. [127] reported the blue shiftof plasmonic band of single gold nanorod when injectingelectrons into it by applying a negative potential. The cor-relation was further utilized to measure the electrochemicalimpedance spectroscopy of single nanoparticles [128]. Theinjection of electrons to a graphene monolayer was alsofound to expand its dimension [129] and to change its optical

conductivity [130]. Both effects can be detected by usingoptical imaging approaches.

4 Single-cell nanosurgery

In a larger scale at mili-meter scale, the cell is the principal,most fundamental unit of life. In 1665, Hooke first used theterm ‘cell’ when observing cork under a microscope [131].Centuries later, Cell Theory was established by Schleiden,Schwann and Virchow, asserting that cells are the primarystructural and functional unit in living organisms, all or-ganisms comprise of one or more cells, and that each celloriginates from a pre-existing cell [132]. As the most basicunit of life, cells each carry out their own functions whichcontribute to the physiology of the organism as a whole,through the expression of genes in each individual cell [133].The common practice of analyzing a population of cells inbulk, however, masks the fundamental differences that existbetween cells, and only gives us an average picture of what isoccurring in healthy and diseased tissues. As result, the in-vestigation of a single cell allows one to explore the in-tracellular and intercellular biochemical process.

4.1 Single-cell isolation techniques

There are many different techniques available in order toisolate single cells for analysis, the most widely-used ap-proaches being Fluorescence Activated Cell Sorting (FACS).FACS depends on the use of fluorescent makers to labeldesired cells, and separates cells from a mixed populationindividually based on their fluorescence. A limitation ofFACS is that as it utilizes a fluid stream of cells, so that tissuesamples need to be dissociated, which could alter their state,and lose contextual information as to where the cells wereisolated from. Laser Capture Microdissection (LCM) offersan alternative, where cells from intact tissue are isolatedunder optical control, retaining spatial information. Although

Figure 7 Schematic illustration of the methodology of optical electrochemical imaging (color online).

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LCM provides spatial context, it does hold its own limita-tions, a major one being the identification of cells of interestamongst a heterogeneous tissue population, which requiresan expert, for example a pathologist. Other disadvantages liein use of staining agents to visualize the tissue and the use offixing agents, which can affect downstream analysis, and theLCM excision itself can damage the cell or cellular com-ponents. It can also be difficult to isolate the cell of intereston its own, without parts of neighbouring cells [134].Fluorescence in-situ hybridization (FISH) is a technique thatemploys fluorescently-labelled nucleic acid sequences todynamically determine the number and location or move-ment of transcripts within single cells. This technique,however, does not work for all transcripts, and is not able toprovide information for as many transcripts at one time as,for example RNA sequencing [135].Microfluidics has had a monumental role in the progress of

single-cell analysis. Microfluidic devices manipulate themovement of liquids within microchannels (on the μmscale), allowing the selective capture of single cells in fluidsuspension. These devices can combine isolation of cellswith lysis and analysis in a single platform [136]. Micro-fluidics can be categorised into either valve, droplet or na-nowell based approaches, which are all compatible withomics analyses [137].One caveat to these devices, however, is that they rely on

the flow of a suspension of cells, which removes spatialinformation as to where the cell of interest was isolated from.Recently, Sarkar et al. developed a microfluidic device thatcan overcome this problem by enabling the selective isola-tion of single cells within a tissue culture. After capture ofthe cell within its tissue environment, it is lysed and bio-chemical analysis is performed, in this case enzymatic as-says. Such a device could therefore be used to correlateintracellular properties of a cell with its spatial context [138].As well as having their own unique drawbacks, all of these

techniques currently used for single cell analysis, aside fromFISH, have the fundamental limitation that they rely on celllysis, and therefore provide only a ‘snapshot’ rather than adynamic analysis of cellular state. There is also a possibilitythat the lysing buffers themselves could influence in-tracellular contents. Furthermore, most of these techniques,aside from LCM and Sarkar et al.’s microfluidic chip [138],remove the cell from its natural environment, losing spatialinformation as to where the cell was isolated.In order to overcome these shortcomings of single cell

analysis approaches, a new field has emerged which employsthe use of nanoscale devices to perform ‘surgery’ on singlecells (Figure 8) [139]. Here, instead of being lysed andanalysed ‘post-mortem’, single cells can be sampled fromcontinuously without affecting their viability or removingthem from their physiological environment. Cellular con-tents are extracted at different time points and subsequently

analyzed, achieving the next level to single cell analysis:spatiotemporal resolution.

4.2 Dielectrophoretic Nanotweezers (DENT)

In 2009, Nawarathna et al. developed a metal-coated AtomicForce Microscopy (AFM) probe capable of extractingmRNA from living cells, which they named the dielec-trophoretic nanotweezer (DENT) [130]. The authors em-ployed a silicon AFM probe with an insulating SiO2 layerdeposited on top, followed by a Platinum layer. Primerscomplementary to specific mRNAs were immobilised ontothe surface, and the AFM probes were inserted in the cellcytoplasm. The application of an AC voltage between thesilicon core and the outer Pt layer generates a dielec-trophoretic force which attracts mRNAs present in the cellcytoplasm which then hybridize to the primers on the AFMprobes. This technique was used to extract the mRNA of thehousekeeping genes β-actin, GADPH and HPRT and thesuccess of the extraction was confirmed by cDNA synthesisfollowed by real-time PCR, and gel electrophoresis. β-actinmRNA was still detected without the application of the al-ternating potential, but applying the potential increased theefficiency of collection. The authors have also shown that theprocedure has non-detrimental effect of cell viability using atrypan blue assay but failed to provide any quantitative in-formation about cell viability.In 2011, the same group demonstrated the extraction of β-

actin mRNA using qPCR and gel electrophoresis. They ad-ditionally demonstrated they were able to extract an onco-gene transcript selectively from cells transfected with thetranscript, in a mixture of both transfected and wild-typecells [140]. In a more recent paper, the group employed anAFM silicon probe coated with SiO2 and a Cr/Au layer. Thisprobe was used to capture DNA molecules in different buf-fers taking advantage of dielectrophoretic trapping [141].The group has since demonstrated the integration of the

technology with microfluidics. The authors have capturedsingle cells in an enclosed PDMS microwell, and use theDENT to penetrate through the PDMS membrane of themicrowell, into the cell, and then employed dielectrophoretictrapping to extract mRNAs [142]. The authors analyzed theexpression of three housekeeping genes from a single liveHeLa cell and quantified their expression levels using RT-qPCR. Furthermore, they were able to analyse the de-pendency on the efficiency of mRNA extraction upon in-creasing the magnitude of the voltage, and concluded thathigher voltages increase the amount of mRNA extracted, dueto higher dielectrophoretic force. The authors carefullyanalyzed cell viability post extraction and they concludedthat the application of a peak-to-peak voltage within therange 1.1-1.5 V resulted in cell viability of around 70% forcells which was similar to the viability of non-probed cells.

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The application of higher voltages up to 1.9V dramaticallydecreased cell viability but the authors did not provide anyquantitative information in their published report.The authors next evaluated the use of the DENT-integrated

microfluidic chip for identifying cancer cells amongst bloodcells, by using a mixture of breast cancer cells and mono-cytes. They trapped single cells, and extracted mRNA anddetected marker transcripts using RT-qPCR. The resultsshowed an mRNA expression ‘fingerprint’ for each cell type,enabling their identification, even though the cells were si-milar in size and visual appearance. These results demon-strate the ability of DENT to selectively extract mRNA forexpression analysis, and indicate its potential applications toidentify specific cell types, for example in a blood sample.In 2019, Nadappuram et al. reported an adaptation of the

dielectrophoretic trapping by employing nanotweezers fab-ricated from dual-carbon nanoelectrodes [143, 144]. The twonanoelectrodes are placed at the tip of a quartz nanopipetteand are separated by a 10-20 nm insulating septum whichenables the generation of extremely high electric field gra-dients (∇|E|2 ≈ 10 28 V2 m−3) that enables the trapping of in-dividual molecules in physiological conditions. The authordemonstrated the extraction of nucleic acid from withinliving cells and also the extraction of one single mitochon-drion from a neuron dendrite (Figure 9). The authors per-formed two biopsies from the same cell and monitored itsviability for approximately 16 hours post-biopsies but theydid not perform any detailed studies on cell viability.

4.3 Fluid-force microscopy (FluidFM)

Another AFM- based approach for single-cell extraction isFluidFM, whereby a cantilever with a nanoscale tip con-taining a microchannel is attached to a pressure controller,allowing pressure driven liquid manipulation. Like DENT,FluidFM retains all the strengths of AFM, in that it can useforce-feedback to precisely position the tip at the surface ofthe cell of interest, and precisely penetrate the cell membrane[145]. Guillame-Gentil et a.l used this technology to extractcontents from living cells through the application of a ne-gative pressure to the FluidFM tip, followed by downstreammolecular analysis [146]. In this study, the pore at the end ofthe modified cantilever tip was 400nm in diameter, and theprobe coated using surface silanization, in order to preventthe extracted contents from depositing on the microchanneland to facilitate the liquid flow.First, extractions were performed from Green Fluorescent

Protein (GFP)-expressing HeLa cells, and the authors de-monstrated successful extraction by measuring the in-tracellular fluorescence decrease upon cytoplasmicaspiration. Next, the authors demonstrated spatially con-trolled extractions, sampling from the nucleus and the cy-toplasm selectively in single cells. The nuclei of the singlecells were labelled with the fluorescent protein mRuby, witha nuclear localisation sequence tag that causes it to be im-ported into the nucleus. Nuclear extractions using theFluidFM probe yielded a reduction in fluorescence intensity,

Figure 8 Schematic showing different methods available to extract cell contents. Reproduced with permission from [139]. Copyright 2017, AAAS (coloronline).

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whereas no decrease in fluorescence was observed uponextracting cytoplasmic contents, indicating mRuby had beenextracted solely from the nucleus. Remarkably, when ex-amining cellular viability post-extraction, the authors foundthey were able to extract up to 4.0 pL from the cytoplasm ofHeLa cells with a cell survival rate of 82%. This represents alarge proportion (around 90%) of the cytoplasm which isable to be extracted with minimal impact on cell viability.Additionally, when performing nuclear extractions, 86% ofcells remained viable after extractions of up to 0.6 pL. Toinvestigate the suitability of the FluidFM approach to beconjugated with mRNA analysis, the authors generatedcDNA copies of transcripts from 2 housekeeping genes andGFP present in cellular extracts, followed by qPCR. Theyanalysed the expression in different cytoplasmic extractstaken from GFP expressing cells, and confirmed the presenceof transcripts for at least one of the genes in 90% of extracts,and all three in two thirds of extracts. Samples taken from thenucleus showed the presence of at least one of the genes inextracts ≥0.7 pL in volume. To build on this work evenfurther, the authors later demonstrated the compatibility ofthe approach with metabolite analysis using matrix-assistedlaser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS) [147]. In this paper, the authors identi-fied 20 cytoplasmic metabolites extracted from HeLa cellsusing FluidFM followed by MALDI TOF mass spectro-

metry. Furthermore, by growing the cells on medium con-taining 13C-glucose, they were able to demonstrate theincorporation of the heavier isotopes into cellular metabo-lites. In 2019, Aramesh et al. used an adaption of FluidFM todemonstrate intracellular detection of biomolecules and ions[148].Taken together these results show the ability to perform

comprehensive structural and biochemical downstreamanalysis on subcellular extracts from cellular compartmentsusing FluidFM with minimal impact on cell viability.

4.4 Nanopipettes

Another method to sample from living cells is based on theincorporation of nanopipettes within a Scanning Ion Con-ductance Microscope (SICM) [149]. SICM is based on anelectrode-fitted nanopipette probe, immersed in a bath con-taining the sample of interest within an electrolyte solution.When a potential is applied between the working electrodeinserted in the nanopipette and the reference electrode im-mersed in the bath, an ion current flows between the two. Asthe nanopipette reaches proximity to the surface of thesample, the ion current will drop as the ion flow will behindered. Because the ion current is therefore distance de-pendent, it can be used as a feedback mechanism to controlthe distance between the tip and the sample, and as thepipette follows the sample surface, the movement of thepipette in the z-direction is measured to recreate the topo-graphy of the sample. SICM is a non-contact method used insolution, it is ideal for high-resolution imaging of live cells,and can recreate cellular topography in real-time [150]. Itsforce-free approach means that it can image delicate andintricate structures, such as neuronal networks and stereocilia[151], without the increased risk of sample damage or de-formation experienced in techniques such as AFM, in whichthe sample encounters nonzero forces [152].Nanopipette fabrication is simple and economical, em-

ploying a laser puller to pull a, typically quartz, capillarygenerating a nanopipette with a tip pore <100 nm in diameter[153]. Nanopipettes can be used to manipulate liquid usingelectrowetting. Here, the nanopipette is filled with an organicsolvent and immersed into the aqueous bath, generating aliquid-liquid interface at its ti A negative potential is appliedbetween the nanopipette electrode and the bath electrode,altering the surface tension, and creating a force large enoughto move the aqueous solution into the pipette. This volumecorrelates to the duration and magnitude of the potentialapplied, and it is possible to manipulate volumes as small as1 attolitre using this technique [154]. One can therefore en-visage a situation whereby the nanopipette filled with or-ganic solution can be inserted into a living cell, harnessingelectrowetting to extract the cytoplasmic contents.Actis et al. demonstrated the use of this technology,

Figure 9 Single mitochondrion extraction with nanotweezers. (a) Sche-matic of the extraction of a single mitochondrion from a neuron dendrite.(b) Fluorescence micrographs after fluorescent staining of the mitochon-dria. (c) Fluorescence micrographs showing (i) targeting of a single mi-tochondrion with the nanotweezers, (ii) trapping of the mitochondrion uponapplication of the AC voltage and (iii) extraction of the mitochondrion(color online).

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whereby they used nanopipettes to sample small volumes, onthe femtoliter scale, from living cells [149]. The procedure isintegrated with SICM, which enables the nanopipette to beprecisely positioned above the cell membrane, followingwhich the membrane is penetrated and cytoplasmic contentsextracted by electrowetting. This process has minimal impacton cell viability, with a survival rate of >70%. The authorsextracted contents from HeLa cells expressing GFP, andgenerated cDNA copies of the RNA extracted, confirmingthe extraction of GFP mRNA using real-time PCR. In ad-dition to this, the compatibility of the nanobiopsy techniquewith next-generation sequencing was illustrated by sequen-cing the cDNA copies of the mRNA extracted from HeLacells. This highlights the robustness of the approach formRNA expression analysis from extractions from singleliving cells.Furthermore, the authors were able to isolate mitochondria

labelled with MitoTracker Green from live human fibroblastcells using the nanobiopsy set-up, and sequenced the mi-tochondrial genomes using next generation sequencing. In2018, Toth et al employed the nanobiopsy platform to in-vestigate mRNA compartmentalisation in neurons [155].The authors aspirated cytoplasmic extracts from differentlocations within the same cell, both in the cell body and theneurites, and employed next-generation sequencing to ana-lyse the extracts. The authors found 1292 genes expressed inthe cell body, and 929 in the neuronal body. It was clear thatthe neurites and cell body had different mRNA expressionprofiles, with an overlap of only around 200 genes, which ishas functional significance. In addition, some species ofmRNAwhich were functionally related localised to the samearea within the cell. Not only this, 178 mRNA transcriptswere detected in neurite nanobiopsies which were low inabundance and have not been detected in bulk analysis in thepast, demonstrating the sensitivity of the technique. Amongthese mRNA species were transcripts which were previouslybelieved to be contained only in the nucleus, and were heredetected in neurites for the first time. Overall, the authorsdemonstrated localisation of mRNA within neurons as wellas proving the ability of the nanobiopsy platform to be usedmultiple times within the same cell and its sensitivity and itscompatibility with next generation RNA sequencing.These studies have only taken advantage of SICM-

controlled positioning of nanopipettes in the z-axis. To buildupon this, Nashimoto et al. integrated two fundamentalcapabilities of nanopipettes; i.e. the ability to aspirate cel-lular contents via electrowetting, and the ability to generate acomplete topographical image of a cell via SICM [156]. Asthe ion current magnitude from a nanopipette filled withorganic solution (dichloroethane) is too small for accuratemapping in the x-y axes, the authors used a dual-barrel na-nopipette with an aqueous-phase barrel serving as an SICMprobe to generate a topographical map of the cell and an

organic phase barrel to aspirate cytoplasmic contents. Thetopographical images of individual mouse fibroblast cellscreated using the aqueous barrel were used to precisely po-sition the pipette, in order to sample from different regionswithin the same cell, and analysis of the extracts providedinformation on mRNA localisation within the cells. Thesame group in 2019 demonstrated that electrowetting-drivencell aspiration can also be performed within tumour spher-oids to study differential gene expression at the surface andat the interior of the spheroid [157].Nanopipettes can also be used under pressure control to

aspirate samples from living single cells. Saha-Shah et al.extracted cytosolic fractions of around 8 nL from Alliumcepa single cells with a nanopipette 600 nm in diameter,followed by analysis by matrix assisted laser desorption io-nisation mass spectrometry (MALDI-MS) and identifiedhexose-oligosaccharides. Furthermore, the technique wasextended to lipid analysis of mouse brain tissue slices andfirst instar Drosophila melanogaster larvae. Altogether thisdemonstrates the versatility of the approach for use in plantcells, tissue samples and complex biological samples, as wellas the compatibility with mass spectrometry identification ofboth hydrophobic and hydrophilic molecules.These results collectively show the wide-reaching cap-

abilities of nanopipettes in use for single-cell analysis stu-dies, being able to extract content from varying cell types,their compatibility with next generation sequencing andmass spectrometry, and the ability to isolate and analyseindividual organelles [158, 159]. Additionally, the conjuga-tion with SICM allows precise positioning of the probe, andmultiple extractions from the same cell in defined locations.

4.5 Nanostraws

A different method of performing extractions from livingcells is using ‘nanostraws’ [160-162]. Here, cells are cul-tured in a dish with a membrane at the base, containinghollow cylindrical tubes, 150 nm in diameter, which projectupwards. A voltage is applied through the nanostraws, whichelectroporates the cells, giving the nanostraws cytoplasmicaccess through the temporary holes created in the membranenear the nanostraw tip, for 2-5 minutes (Figure 10). Thisallows intracellular contents to travel through the nanos-traws, by diffusion, into a reservoir containing buffer solu-tion below, which can then be collected and analyzed. Assuch, the extracts from each cell are collected in the samebath and analysed together. The sampling area can be definedusing photolithography, enabling sampling from regions<1μm to mms in size.To illustrate the efficacy of this approach, Cao et al. per-

iodically sampled from an area of 38 GFP and Red Fluor-escent Protein (RFP)-expressing CHO cells and verifiedsuccessful extraction by measuring the fluorescence intensity

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of the extracts using fluorescence microscopy. The samplingarea was then decreased in order to sample from a single cell,and the amount of RFP extracted over a 4-day period wasmeasured pre- and post-transfection with an RFP plasmid atday 2. The authors found that the increase in the RFP contentof the extracts correlated with the increase in RFP expressionby the cell. The resulting cell viability was the highestamongst all nanosurgery techniques with over 95% survivalrate, showing the ability of this technique to sample fromcells longitudinally and non-destructively.The authors further validated the nanostraw platform as a

longitudinal live cell-sampling method by sampling fromhuman induced pluripotent stem cell (hiSPC)-derived car-diomyocytes and astrocytes over a 5-day period. They usedan enzyme-linked immunosorbent assay (ELISA) to detectthe heat shock protein 27 (HSP27) in extracts following theincubation of cells at 44 °C for 30 minutes. An upregulationof HSP27 expression was observed after the heat shock. Thiswas performed on an area containing 100 000 cells, in orderto be compatible with the ELISA sensitivity. Finally, mRNA

was extracted from hiSPC-derived cardiomyocytes, and theexpression of a selected set of mRNAs was analyzed, com-pared to a lysed cell. All mRNAs detected in the lysed cellwere also present in the nanostraw-derived extract. Thisprocess required 15-20 cells for sufficient mRNA analysis,however, there is the possibility for single-cell mRNA ex-pression detection to be performed in the future as mRNAanalysis techniques become more sensitive. Furthermore,contrary to other techniques outlined in this review, nanos-traws allows parallel extraction from cells, rather than con-secutive sampling.

4.6 Nanotube endoscopes

Singhal et al. have demonstrated that a carbon nanotube thatbe inserted within the tip of a micropipette to comprise ananoscale endoscope able to penetrate a cell membrane toperform intracellular analysis [163]. The authors demon-strate that the carbon nanotube can be decorated with goldnanoparticles and Surface Enhanced Raman Spectroscopy(SERS) spectra can be acquired within the intracellularspace. The authors also hint at the potential for single-cellsurgery by showing some preliminary data on single orga-nelle probing but do not perform any downstream molecularbiology analysis. Similarly, Yan et al. [164] attached a na-nowire on the tip of a tapered optical fibre to comprise ananowire endoscope. The authors employed it for in-tracellular cargo delivery and for optical interrogation ofintracellular compartments with nanoscale resolution. Al-though both these nanoendoscope techniques have not beenused for extraction of cellular contents they both hold greatpromise for simultaneous detection and extraction of in-tracellular material.

5 In vivo electrochemistry in brain research:challenges and solutions

To explore the molecular mechanism and chemical in-formation of brain function is one of the important frontiersof modern neuroscience, and brings both great challenge andopportunity to chemists. While we are still in the learningphase in many aspects of brain chemistry, the electro-analytical community is certainly making significant ad-vances down this path, with a broad range of micro-sized andchemically engineered electrodes that have proven to behighly effective in measuring rapid chemical changes in liveanimal brains.

5.1 Overcoming the in vivo selectivity challenge bymodulating the interfacial electron transfer

There are many types of chemical substances exist in the

Figure 10 Schematic of the nanostraws technology. (a,b) This samplingtechnique is based on a polycarbonate membrane with protruding 150-nmdiameter nanostraws supported on a cell-culture dish. Sampling is triggeredby the application of short voltage pulses that temporarily electroporate thecells, allowing cellular content to diffuse through the NS and into theunderlying fluidic reservoir (highlighted in pink in a)), which is thenanalysed using fluorescence imaging, ELISA, or qPCR. (c,d) SEM imagesof the 150 nm diameter NS (c) and the 200 × 200 μm2 active samplingregion (d). Cells outside this window are unaffected by the sampling pro-cess. (e,f) SEM images of cells cultured on a 200 × 200 μm2 active sam-pling region containing 42 cells (e) and a 30 × 30 μm2 sampling region usedto isolate and sample from a single cell (f) (color online).

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brain, which are unevenly distributed, and transformed in anexquisitely ordered manner. It therefore remains as one of themost challenging issue to selectively measure the neuro-chemical in such a complicated environment, particularly ina live brain. The traditional approaches for sensitive in vivoelectrochemical analysis rely on the development of newelectrochemical technologies that discriminate neurochem-icals based on their own voltage-dependent oxidation andreduction processes. For instance, fast scan cyclic voltam-metry (FSCV) as a typical technology has garnered con-siderable interest in neurochemical research community. Atthe ultrafast scanning speed, neurochemicals of differentelectron transfer kinetics can become distinguished as theirredox peaks shift to distinct positions. By virtue of its se-lectivity and millisecond temporal resolution, over the past20 years, FSCV has been extensively adopted to study thedynamics of neurochemicals, such as dopamine and ser-otonin in live animals. However, being a background-sub-tracted method, FSCV inherently requires complicated datacollection, analysis and statistic models to improve the signalresolution. Moreover, great efforts have also been con-tributed to extend its suitability to neurochemicals that areless electroactive, which greatly restrict FSCV from thewider in vivo analysis applications.We began to work on selective in vivo analysis strategy and

principles since 2002, and focus on designing new chemicalapproaches to modify electrode surface, and to modulate theelectron transfer at its interface, enabling in vivo electro-chemical monitoring of brain chemistry with excellent se-lectivity. The very first example in this line wasdemonstrated by using carbon nanotubes (CNTs) to constructelectrode/electrolyte interface for selective electro-oxidationand in vivo analysis of Vitamin C (VC, also named as as-corbic acid, AA) [165, 166]. We found that VC oxidationcan be greatly accelerated on heat-treated CNTs, primarilyowing to the presence of edge plane-like carbon and ex-cellent electronic properties of CNTs [165-170]. More im-portantly, the CNTs-facilitated electron transfer works nicelywhen it was integrated with in vivo analytical system, al-lowing selective determination of VC against interferingneurochemical species in the central nervous system (CNS)(Figure 11).Venton et al. reported the use of CNTs modified carbon

fiber microelectrodes for monitoring simultaneous release ofdopamine and serotonin in vivo. To further improve theelectrocatalytic performance of CNTs, Zhang et al. en-gineered tunable defects and oxygen-containing groups inCNTs which exhibited a greatly facilitated VC oxidation at alow potential, allowing in vivo detection of VC levels in ratbrains under both physiological and pathological conditions(Figure 12) [169]. Besides CNTs, carbon black also serves asan excellent electrode material owing to its merits of highconductivity, large surface area and low cost. For example,

by coating nano-composites of carbon black and mediatoronto electrode surface, Cheng et al. constructed a ratiometricelectrochemical sensor for selective VC measurement in livebrains [167].The successful modulation of electron transfer by tuning

the surface chemistry of electrode using CNTs further in-spired our studies of other carbon nanomaterials that featuredistinct chemical and physico-chemical properties, such asgraphdiyne. We found that oxidized graphdiyne (GDO),which contains a large degree of electron-withdrawingacetylenic bonds, exhibiting an ultrafast humidity responsewith an unprecedented response speed (ca. 7 ms) [170]. Suchchemical attributes along with its electrochemical propertiesenable us to fabricate a GDO-based humidity sensor withexcellent selectivity against other types of gas molecules,and allow accurately monitoring of the respiration ratechange in living systems. Inspired by the electrochemicalattributes of carbon nanomaterials, we reason that the single-atom catalyst (SAC), featuring atomic active sites that aredispersed in ordered porous N-doping carbon matrix, couldbe superior in catalyzing the electrochemical process ofphysiologically relevant chemicals. As demonstrated veryrecently by our group, we found that a cobalt-based single-atom catalyst (Co-SAC) can catalyze the electrochemicaloxidation of hydrogen peroxide (H2O2) at a low potential ofca. +0.05 V(vs. Ag/AgCl), therefore, allowing for the furtherdevelopment of an oxidase-based biosensor for selectivemonitoring of glucose in rat brain.Other than using synthetic nanomaterials to tune the in-

terfacial chemistry of electrode for selective in vivo analysis,

Figure 11 (a) Schematic diagram of analytical system for selective de-termination of VC in rat brain. Reprinted (adapted) with permission fromref. [166] Copyright (2019). (b) Typical cyclic voltammograms (CVs)obtained at heat-treated CNTs modified-electrodes in the presence (solidlines) and the absence (dash lines) of VC. (c) Online measurement of VC inthe brains of ischemia rats (black line) and shame-operated rats (red line).Reprinted (adapted) with permission from [165] (color online).

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enzyme-modified electrodes remain particularly attractive indeveloping sensors with high selectivity. Generally, flavinadenine dinucleotide (FAD)-containing oxidases and dehy-drogenases are two types of enzymes that have been widelyemployed to construct electrochemical biosensors, in whichthe oxidase-based biosensors are more popular for in vivoanalysis. This is because their ease in fabrication with lessinterference from hydrogen peroxide, one of the mostabundant, small-molecule oxidants exist in the cerebralsystems. Nevertheless, the requirement of using mediators,for example ferricyanide/ ferrocyanide (Fe(CN)6

3-/4-) whoseredox potential generally overlaps with those endogenouslyexisting neurochemicals in the cerebral systems makes themfar from ideal for in vivo neurochemical measurements. Torealize the negative shift of the redox potential of Fe(CN)6

3-/4-,we have developed the modification of an imidazolium-based polymer (Pim) onto electrode, controlling the different

adsorption ability of Fe(CN)63- and Fe(CN)6

4- onto electrodesurface [168]. This strategy has led to a negative shift of theredox potential of the surface-confined redox mediator.Using glucose oxidase (GOD) as the model recognitionunits, we have demonstrated the validity of the ferricyanide-based second-generation biosensors for selective in vivoglucose measurements (Figure 13).Given the great variation of pH and oxygen levels involved

in most of the physiological and pathological processes, asensor with high tolerance against oxygen and pH would bethe most desirable for in vivo measurement. Since the de-hydrogenase-based catalytic chemical oxidation of glucoseand lactate is oxygen-independent, we adopted the dehy-drogenase as the recognition element on the electrode surfaceand developed dehydrogenases based electrochemical sys-tem that allows selectivity monitoring of glucose and lactatein brain with minimal interference from pH and oxygen

Figure 13 (a) Typical CVs obtained at multiwalled CNTs (MWNTs) modified electrode (black line) and Fe(CN)63-/Pim/MWNT-modified electrode (red

line). (b) Typical amperometric response of microdialysate sampled continuously from the striatum of guinea pigs. Reprinted from [168] with permissions(color online).

Figure 12 (a) The absorption of AA on defect-containing CNTs and differential pulse voltammetry (DPV) records obtained in the striatum of normal rat(blue line) and rat model of Alzheimer’s Disease (red line). Reprinted (adapted) with permission from ref. [169] Copyright (2017); (b) Schematic illustrationof ratiometric electrochemical sensor for measuring AA in live brains. Reprinted (adapted) with permission from ref. [167] Copyright (2015); (c) Schematicillustration of Graphdiyne oxide (GDO) and current responds of both GDO-based (red line) and Graphene oxide (GO)-based (blue line) sensor toward humidair. Reprinted (adapted) with permission from ref. [170] Copyright (2017); (d) Schematic illustration of Co-SAC and typical amperometric responses from ratstriatum under normal state and after injection of insulin. Reprinted (adapted) with permission from ref. [166] (color online).

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[171].Taken together, the use of synthetic nanomaterials or

naturally existing enzymes as electrocatalysts to modulatethe interfacial electron transfer of electrodes have proven tobe efficient and contributed significantly in solving the long-lasting challenge in the selective in vivo analysis of neuro-chemicals.

5.2 Overcoming the in vivo stability challenge by de-veloping the Galvanic Redox Potentiometric (GRP)sensors

Amperometry permits selective and sub-second in vivomeasurements of neurochemicals, allowing their wide use toelucidate the physiopathological roles of neurochemicals inanimals. However, one of the main drawbacks associatedwith amperometry is the instability of electrode in vivo,which results in a dramatic drop in current due to the elec-trode passivation by nonspecific adsorption of proteins inbiological fluids. These issues affect the relationship be-tween the current and the concentration, restricting a stablein vivo measurement. In this regard, determining the basallevels and monitoring the dynamics of neurochemicals re-quire the in vivo analysis to be sensitive yet stable in thecomplex environment. In view of this challenge, potentio-metric measurements based on Nernst equation is gaining inprominence because they are less affected by the surface areaof working electrodes. One of the best-known potentiometricsensors is ion-selective electrode (ISE), which is widelyutilized to determine ion concentration by measuring trans-membrane potential. Being implanted in rat brains, ISEs hasextended the applications for in vivo measurement of K+, H+

and Ca2+ [172, 173]. Recently, a fluorescent nanosensor hasalso been used for in vivo monitoring of extracellular K+

levels in the brain [174].In addition to ions, we have designed the very first open-

circuit potentiometric-based sensor, termed galvanic redoxpotentiometric (GRP) sensor, for measuring the moleculardynamics of neurochemicals in live brain with satisfying

selectivity, unaltered sensitivity by electrode fouling, andhigh reliability [175]. The direct electron transfer (DET) oflaccase on CNT-modified electrodes allows the constructionof Galvanic cell, through which the oxygen can be reduced ata high potential where the thermodynamics allowed, pro-viding the foundation for the establishment of GRP method.Moreover, we observed a dramatic enhancement of oxygenreduction current response (by ca. 600% at maximum) uponethanol-assisted immobilization at carbon nanotube electro-des, achieving both high potential and high current si-multaneously, which provides the possibility of using laccaseelectrode as reference electrode of GRP, The GRP sensorovercomes many scientific bottlenecks, such as the realiza-tion of the direct electron transfer of laccase and constructionof Galvanic cell, successfully transforming the detection ofredox active substances from traditional current-basedmethod to potential-based approach, providing new elec-trochemical principles to solve inherent stability issues incurrent in vivo analysis systems (Figure 14).While the GRP sensor has been demonstrated to be very

attractive for stable in vivo analysis, the selectivity remains abig concern. The use of enzyme-based biosensing strategywould pave an effective way to this problem. Very recently,we have used recombinant Ferredoxin-dependent glutamatesynthase (Fd-GltS) from cyanobacteria as an enzymaticbioelectrocatalyst to develop a new electrochemical bio-sensor for glutamate detection. Particularly, bioelec-trocatalytic oxidation of glutamate by Fd-GltS was oxygen-independent, overcoming the challenges that associated withoxidases or dehydrogenase-based bioelectronic sensors,which are oxygen dependent, and the requirement of co-enzymes, respectively. Moreover, as the redox centers of Fd-GltS are close to enzyme surface, making it prone to realizedirect electron transfer with the electrode. Taken together, itis favorable to use GRP principles to develop new-genera-tion of electrochemical sensors for in vivo analysis of glu-tamate.With all above efforts, we further extended the GRP

strategy for in vivo monitoring of VC, as a proof-of-concept

Figure 14 (a) Schematic illustration of in vivo AA monitoring by using GRP sensor. (b) Calibration curves of the GRP sensor before (black line) and after(red line) BSA adsorption. (c) In vivo AA monitoring during global cerebral ischemia (red arrow) and reperfusion (blue arrow). Reprinted (adapted) withpermission from ref. [165] (color online).

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study. The GRP sensor is constructed on a self-driven gal-vanic cell, where VC is spontaneously oxidized at the in-dicating single-walled carbon nanotube modified electrode(SWNT-CFE), while oxygen reduced at the laccase-modifiedreference CFE (Lac-CFE). The as-designed sensor shows ahigh selectivity to VC in the presence of coexisting elec-troactive neurochemicals. It further enables the measurementof the basal level of cortical VC in live rat brain (230 ±40 μM) and its fluctuation during ischemia/reperfusion. It isworth of note that the sensitivity of GRP sensor is tolerant toprotein adsorption or electrode fouling, offering a prominentmethod for in vivo, real-time, stable and reliable analysis ofbrain neurochemistry.

5.3 Neurobiology studies enabled by new in vivo ana-lytical methods

Monitoring the dynamic change of neurochemicals in livinganimals can provide important information for understandingthe molecular mechanism of CNS, as well as the cause ofneurodegenerative disorders. The key challenges associatedwith brain chemistry research lie in the deciphering of neu-rochemical signaling in terms of its chemical, spatial, andtemporal information. In this regard, in vivo electrochemicalmonitoring of neurochemical is of great potency for above-mentioned neurobiological studies. For example, our in vivoelectrochemical systems, established either through mod-ulating the electron transfer by engineering of interfacialchemistry or outputting signals using potential-based GRPmethod, have been demonstrated to be satisfyingly selectiveand stable in monitoring of neurochemical fluctuation inliving brain of animals with high temporal and spatial re-

solution. Specifically, being able to deploy micro-sizedsensors into specific region of brain, has been the corner-stone for the realization of in situ monitoring of a greatvariety of neurochemicals, including VC [176], dopamine[177], glutamic acid [178], oxygen [179], glucose [180],lactic acid [181], hypoxanthine [182], ATP [183], and H2S[184], offering new analytical approaches for advancing ourunderstanding of the brain science.Particular exciting is that with our in vivo analysis system,

we demonstrated that VC might exhibit some unexploredroles in certain physiological events associated with braindiseases. For example, we found that VC can be released inthe form of nanoscale vesicles, the main pathway that iscommonly preserved in the regulated secretion of neuro-transmitters by neurons (Figure 15(b)) [185]. Moreover, weobserved for the first time that the propagative nature of VCrelease following the spreading diffusion process, high-lighting its importance as a neuromodulator in mediatingneurotransmission (Figure 15(a)) [186]. Furthermore,through our long-term collaboration with Peking UniversityThird Hospital, which specializes in designing, establishingand evaluation of clinical relevant disease models, we de-monstrated that VC plays more important roles other thanjust as free radical scavengers for neuro-protection in theevents of tinnitus, olfactory dysfunction and Vertigo [187].Most importantly, the approach for in vivo analysis of

glucose, lactic acid, ATP, glutamic acid, pH, calcium ionsand other neurochemicals, has been demonstrated to beprofoundly effective by some domestic institutes and hos-pitals in their own independent research. This interest inusing the in vivo electrochemical sensors can be attributed totheir excellent selectivity and stability, two essential pre-

Figure 15 (a) Typical current recordings upon electrical stimulations (red bars), insert, schematic illustration of experiment setup (top) and VC release indifferent detection sites followed by electrical stimulations (red bars) (down). Reprinted (adapted) with permission from ref. [186] Copyright (2019); (b)Single-cell amperometric recordings of vesicular VC release by K+-stimulated exocytosis. Reprinted (adapted) with permission from ref. [185] (coloronline).

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requisites for reliably capturing and further interpreting thedynamics of neurochemicals in the complicated centralnervous system. The application of our in vivo electro-chemistry based neurochemical sensors has provided a solidexperimental foundation for exploring molecular mechan-isms of brain function and highlighting the key role andbright future of analytical sciences in brain research.

6 Towards precision electrochemical measure-ments

The challenge in developing meaningful and fundamentallyaccurate descriptions of complex electrochemical mechan-isms, e.g., the multi-step reduction of O2, is in understandingthe interfacial structure with a high degree of precision, andby improving the temporal and spatial resolution of methodsto interrogate reactions.Over the past decades, electrode fabrication in the micro-

and nano-domains has allowed studies of single entitiesranging from individual molecules to single nanobubbles,pushing analytical electrochemistry to the regime of unitsensitivity with unprecedented precision at the single-entityscale [1, 188, 189]. Nanoelectrodes offer significant ad-vantages over macro and microelectrode geometries inprobing chemical and kinetic properties at the individualmolecule or nanoparticle scale. Often this is done by cou-pling extremely small background double-layer capacitancevalues with near-instantaneous steady-state concentrationprofiles to offer the spatiotemporal resolution necessary toprobe electron transfer kinetics in the nanosecond regime[190-192]. In recent years, the employment of nanoelec-

trodes in electrochemical microscopy techniques has becomeincreasingly important to study a wide variety of differentreactions [193-195], and to elucidate the localized structure-activity in a wide range of electrochemically active nano-materials (e.g., nanotubes, graphite and graphene, nano-particles, and materials for energy storage) [195, 196]. Theseprecision measurements pave a new way to explore me-chanisms of complex chemical process, expanding analyticalapplications for electrochemistry.

6.1 Counting molecules in nanobubbles

Gas generation with phase formation is important in bothfundamental studies of nucleation and many electrocatalyticprocesses [197, 198]. Nucleation, the first step of phaseformation, takes place in a very short time at nanometerlength scales. While classical nucleation theory (CNT) de-scribes average rates of nucleation [199], direct and precisemeasurements of the nucleation of single gas nuclei arebarely reported [200-202]. Therefore, a detailed quantitativemeasurement of the behavior of nanobubbles on the elec-trode surface is of great importance.A single-entity approach was recently developed to ana-

lyze the nucleation of single nanobubbles from precisemeasurements at nanoelectrodes [203]. Taking H2 bubbleformation as an example (Figure 16(a)): during the voltam-metric scan, when hydrogen evolution reaction occurs, theconcentration of dissolved H2 near the electrode surface in-creases as the current increases. When the concentration ofH2 is sufficiently high, a H2 bubble nucleates stochasticallyfrom the supersaturated solution, and rapidly grows and

Figure 16 (a) Schematic of the nucleation and growth of a single H2 nanobubble on a Pt nanoelectrode: ① H2 produced by proton reduction remainsdissolved. ② A critically-sized nucleus forms. ③ A single stable bubble covers nearly the entire electrode. (b) Cyclic voltammogram showing bubbleformation at a 10 nm Pt disk electrode in 1.0 M H2SO4 (10 cycles shown); numbers relate to stages in part a. (c) histogram of nucleation peak currents (iH 2 nb

p )from 400 voltammetric cycles and theoretical fit using J0 = 6.3 × 1012 s-1 and θ = 150°. (d) Schematic of the critical nucleus for H2 bubble derived fromanalysis of iH 2 nb

p in the voltammograms in (b). Reproduced, with modification, from ref. [203] copyright 2019 by the American Chemical Society (coloronline).

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covers the electrode surface. This coverage blocks the activeelectrode surface, causing a sudden decrease of the current,resulting in a peak current, i H nb

p2, in the voltammogram. The

peak current occurs at the moment when H2 bubble nucleatesand can be used to calculate the concentration of H2 requiredfor nucleation.Further information of bubble nucleation rate and prop-

erties of the critical nucleus can be obtained by analyzing thedistribution of i H nb

p2. The experimentally obtained i H nb

p2

inmultiple voltammetric cycles is highly reproducible (∼3%relative deviation, see Figure 16(b) and (c)), even thoughnucleation of a single bubble is stochastic, as has been de-monstrated in previous induction time measurements forsingle bubble nucleation [204]. This seemingly deterministicbehavior of i H nb

p2

can be explained by applying classicalnucleation theory and statistics (survival analysis) to thevoltammetric response. The experimental distribution ofi H nb

p2

in hundreds of voltammetric cycles can be well fittedby the theory (Figure 16(c)) using two free variables inclassical nucleation theory; the pre-exponential factor J0, andthe contact angle of the bubble nucleus, θ.With the theoretical analysis, a voltammetric approach was

developed to measure parameters of the nanoscopic bubblenuclei by analyzing the small variation, i.e., the “noise”, inthe peak current. The distribution of the peak current fornucleation is precisely measured in multiple voltammetriccycles and the distribution of this value is fit by theory usingcombination of J0 and θ. It is found that a H2 bubble nucleushas a contact angle of 150° (Figure 16(d)). Together with thepressure inside the bubble nuclei, also calculated using thevoltammogram, the number of H2 molecules in a H2 bubblenucleus is ~ 50. This voltammetric approach can also beapplied to the nucleation of bubble of other gas types, in-cluding N2, O2, and CO2. Such an approach demonstrates thepower of single entity electrochemistry.

6.2 Sub-nanometer resolved particle size measure-ments

Achieving precise information about single entities, requiresone to minimize the influence of extraneous sources of noise.While careful choice/design of electronic circuitry, appro-priate grounding, and shielding from electromagnetic ra-diation (e.g., through using a Faraday cage), are an advisablefirst course of action, in order to minimize the influence ofelectrical noise, further improvements require additionalstrategies. Repeated measurements of the same quantity arecommonly performed in analytical chemistry, as a means toimprove measurement precision. While repeated measure-ments of individual nanoscale entities may present newmeasurement challenges, in addition to improved precision,

they can provide additional information, e.g., through char-acterizing the distribution of an inherently stochastic quan-tity, or by observing processes that would be averaged overin an ensemble measurement.As described in Section 2, in resistive pulse sensing, the

ion current between two electrodes, one inside an electrolyte-filled nanopipette/nanopore, the other in bulk solution (Fig-ure 17(a)), is used to characterize a single nanoscale entity asit passes through the pipette/pore orifice. The momentarydrop in current that occurs as an entity translocates providesinformation on its size, shape and more [28]. Using com-puter-controlled instrumentation, which facilitates repeatedresistive pulse measurements of individual nanoparticles insolution, an augmented resistive pulse sensing method wasdeveloped, which allows measurements of individual parti-cle properties with enhanced precision [205], and facilitatesnew measurements [206].As shown schematically in Figure 17(a), a nanoparticle

passing through the orifice of the nanopipette, is detected bya resistive pulse and, after a predetermined (~ms) delay, thedirection of the forces acting upon the nanoparticle are au-tomatically reversed [208]. In the example shown, reversingthe polarity of the applied potential (Figure 17(b)) reversesthe net force on the particle. In this case, the forces arise fromelectroosmotic flow and electrophoresis, but altering thehydrostatic pressure across the pore can achieve a similarresult [205]. The reversed forces drive the particle backthrough the orifice, whereby another resistive pulse is de-tected, and the wait-switch-detect cycle repeated. Figure 17(b)/(c) shows the potential/current-time traces correspondingto four resistive pulses from the same particle passing out-in-out-in, which were taken from the larger dataset shown onthe right hand portion.

Figure 17 Multipass resistive pulse sensing of a single nanoparticle. (a)Schematic of measuring a single nanoparticle passing forth and backthrough a nanopipette via voltage switching. (b) Voltage-time, V-t, and (c)current-time, i-t trace of four resistive pulses from one nanoparticle (out-in-out-in sequence). Right-hand side plots are the V-t and i-t traces on anexpanded time range. Adapted from ref. [207], copyright American Che-mical Society (color online).

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The exclusion of conductive electrolyte, equal in volumeto the particle typically causes the largest contribution to theamplitude of the resistive pulse; however, variability in theamplitude arises from variability in the position of the par-ticle as it translocates, relative to the pore axis [209]. Re-peated measurements of the same particle can eliminate (andcharacterize) this variation, thus with sufficient measure-ments of the same particle (>10,000 demonstrated [207]) thevolume of a single particle can be characterized with es-sentially arbitrary precision [205, 207].When non-spherical objects translocate, the resistive pulse

reflects both the orientation and volume of the object. Forexample, a nanorod with its long axis parallel to the axis ofthe pore gives a lower resistive pulse amplitude compared tothat of a sphere of equal volume, whereas when the axes areperpendicular, this relationship is reversed. Moreover, if theorientation of a particle charges during translocation, thelocal maxima and minima are observed in the otherwisesmooth resistive pulse. Analysis of the time between peaksinforms on the tumbling rate, which we related to the rodshape through equations of rotational diffusion [210]. Whilethe amplitude of the resistive pulse is typically dominated bythe volume of entity that is translocated, the shape of theresistive pulse contains additional information, including onthe surface charge [211], bipolar electrochemistry [212], andthe mechanical properties of soft entities [213].The combination of nanoparticles and nanopipettes ex-

tends beyond resistive pulse sensing [214]. When a pipette isplaced close to an electrode resistive pulse characterizationcan be combined with delivery of a particle to a surface,allowing for correlated electrochemical characterization andprobing of the dynamics of particles [215-217]. While suchtandem measurements have not been combined with theextra precision of multipass measurements, it seems only amatter of time before they are.

6.3 Towards electrodes of molecular dimensions

Precision measurements based on nanoelectrodes have beenintegral in revealing physicochemical properties at singlemolecules and nanoparticles; however, reproducible and re-presentative measurements at this length scale necessitaterobust nanoelectrode fabrication techniques to extractmeaningful insight. Since 2001, with the seminal work ofKatemann and Schuhmann describing platinum disk nanoe-lectrodes fabricated via laser pulling of quartz-sealed Ptmicrowires and subsequent focused ion beam (FIB) millingto expose the Pt surface [218], robust bench-top techniquesto generate a wide range of nanoelectrode geometries havebeen described including inlaid disks [192], nanopore [191],nanogap [219], nanoelectrode arrays [220], and nanoparticleelectrodes [221]. These techniques have been extended be-yond Pt, to generate Au [222], Ag [102], Cu [223], and C

nanoelectrodes [224, 225] offering a wide range of electrodematerials suitable for fundamental electrochemical studies ofboth inner- and outer-sphere electron transfer processes.While these nanoelectrodes offer unparalleled electro-chemical sensitivity at the single entity level, the stability ofnanoelectrodes following fabrication and during repeatedmeasurements remains a challenge in precise single entitystudies [1]. As highlighted in Amemiya’s 2013 work onelectrostatic discharge damage to Pt nanoelectrode surfaces,extensive care must be taken to ensure the geometry of ananoelectrode has not been perturbed prior to or during anelectrochemical measurement [226]. While electrode foulingvia organic molecule adsorption can be countered withplasma treatment [227] or FIB milling to re-expose the na-noelectrode, operator handling of these electrodes during thecleaning process introduces another opportunity for elec-trode fouling. FIB milling of laser-pulled quartz-sealed Ptwire nanoelectrodes remains the preferred method for fab-ricating low RG nanoelectrodes (RG is Rg/a, where Rg is theradius of the insulating glass and a is the radius of the mi-crowire) for nano-SECM or in-vivo single cell measure-ments. However, high RG nanoelectrodes can be generatedby sealing either a) cyanide etched Pt or Au wires or b)pulled quartz-sealed Pt nanoelectrodes in a larger capillaryand mechanically polishing the electrode on an aluminapolishing pad with an electrical continuity probe to indicatenanoelectrode surface exposure [192]. Pt nanoelectrodesfabricated via laser-pulling offer stable voltammetric re-sponses for ferrocene oxidation in acetonitrile after CV cy-cling between +800 mV and -800 mV vs. Ag/AgCl (Figure18a). However, pulled and etched nanoelectrodes are oftenrecessed following polishing or exposure to solvent condi-tions [219, 228], steady-state voltammetric response with asmaller apparent electrode radii when assuming an inlaiddisk geometry, a feature that cannot be detected or char-acterized solely by cyclic voltammetry.Analytical solutions for diffusion in a recessed nanoelec-

trode can be used to ensure that measurements at theseelectrodes is physically meaningful, however, the apparentradius will be consistently underreported if an inlaid diskgeometry is assumed without verification via SEM, TEM, orAFM. Additionally, the geometry of an otherwise wellcharacterized electrode can change rapidly following ex-posure to new conditions, as emphasized in Figure 18(b)demonstrating the change in steady-state current for ferro-cene oxidation in acetonitrile after the electrode was dippedin deionized water for less than five seconds, correspondingto an apparent recession of ~85 nm. Furthermore, SEMcharacterization of these nanoelectrodes suggests that theexposed Pt surface is sensitive to handling and exposure tosolvent under grounded conditions, as illustrated by thechange in topography and equivalent radii in Figure 18(c-e).As the stability of nanoelectrodes under a wide range of

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solvent conditions, namely high cathodic potentials andaqueous solutions, have not yet been thoroughly examined,extensive care should be taken to verify nanoelectrode fi-delity through the course of measurements, particularly at thesingle entity domain where variations in analyte flux profilesare accentuated between an ideal and a geometrically un-certain electrode, or when measuring electron transfer ki-netics [229].

6.4 Electron transfer at individual geometrical lineinterfaces

Coupled electron- and phase-transfer (CEPhT) reactions playa key role in many electrochemical systems, especiallywithin energy conversion and storage technology. For ex-ample, during the intercalation of Li+ in lithium-ion batteries,the Li+ must cross from the liquid electrolyte phase into theelectrode material concurrently with an electron transferprocess, where the electrode material is oxidized or reduced.In a similar fashion, CEPhT reactions are also observedduring electrodeposition, where the redox species transfersfrom a liquid phase to a solid phase while being oxidized orreduced. Furthermore, CEPhT reactions are essential in

multiphase organic electrosynthesis [230], which has beenshown to be an environmentally friendly, economical, andsafe alternative to homogeneous organic electrosynthesis.Multiphase electrosynthesis relies on the use of three im-miscible phases, such as an electrode placed in an oil-in-water emulsion. These multiphase syntheses generally in-volve a CEPhT reaction, where either the reactant, product,intermediate, mediator or catalyst will undergo a phase-transfer coupled to an electron-transfer. In these examples,the three-phase interface is generally not geometrically welldefined, making the mechanism of the reaction difficult toprobe. For this reason, fundamental understanding of CEPhTreactions has been limited. To obtain insights into the ki-netics, energetics, and mechanism involved in CEPhT reac-tions, a cylindrical wire electrode was placed across theinterface of two immiscible liquids, creating a geometricallywell-defined, three-phase interface of nanometer thickness[231, 232]. This system allows for a more precise study ofCEPhT reactions that can be computationally modeled withrelative ease.The schematic in Figure 19 shows a Pt-Ir cylindrical wire

across a 1,2-dichloroethane (DCE)/H2O interface, whereboth liquid phases contained electrolyte, and the redox spe-cies ferrocene (Fc) was initially present only in the DCE. Asshown in Figure 19, the CEPhT reaction can happen by threemechanisms: the electron transfer step either preceding (case1) or following (case 2) the phase transfer, or in a concertedmechanism, where the electron-transfer occurs concurrentlywith the phase transfer and no stable population of reactionintermediates is produced (case 3). Voltammetric character-ization can experimentally distinguish between these threepossible CEPhT mechanisms. Figure 20(a) shows two con-trol experiments where the Pt-Ir wire was placed in the H2Ophase (red) and the DCE phase (black). No faradaic current isobserved with the Pt-Ir wire in the H2O phase as Fc is notsoluble enough in H2O to be electrochemically observed.When the Pt-Ir wire is placed in the DCE phase a voltam-metric wave is seen corresponding to the oxidation and re-duction of Fc present in that phase. Figure 20(b) shows thevoltammetric response with the Pt-Ir wire placed across theDCE/H2O interface. A similar response to the black curve inFigure 20(a) is observed, but two extra peaks are seen atlower potentials. After the initial oxidation of Fc in the DCEphase at ~0.63 V vs Ag/AgCl, the charged Fc product (Fc+)produced near the three-phase interface transfers into theH2O phase, due to its higher solubility in that phase. On thereverse scan of the first cycle, two reduction peaks are ob-served at 0.53 V and 0.28 V for the reduction of Fc+ in DCEand H2O, respectively. On the second cycle, Fc is present inboth phases and oxidative peak currents are seen for thereaction in H2O at 0.38 V and DCE at 0.63 V. Continuouscycling produces a steady-state response qualitatively similarto the second cycle in Figure 20(b).

Figure 18 Nanoelectrode Stability. (a) Cyclic voltammogram of rapp52 nm inlaid disk Pt nanoelectrode in 5 mM ferrocene in acetonitrile(black) with subsequent recession by ~85 nm following exposure to aqu-eous conditions, resulting in voltammogram corresponding to an rapp18 nmPt nanoelectrode. (b) Cyclic voltammogram of rapp150 nm inlaid disk Ptnanoelectrode in 5 mM ferrocene in acetonitrile cycling between 0 to 0.8 Vvs. Ag/AgCl following cycling between 0.8 V and cathodic limit for 10cycles, demonstrating minimal variation in apparent radius between sub-sequent voltammetric cycling. (c) SEM characterization of inlaid disk Ptnanoelectrode with an equivalent radius of 238 nm with subsequent re-mounting in SEM (d) and in situ exposure to 100 µL acetonitrile (e) de-monstrating significant variations in NP topography and the emergence ofcharacteristic nanoelectrode recession following electron beam exposureunder grounded conditions (color online).

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Further experiments using this setup, determined theCEPhT reaction to be highly dependent on the three-phaseinterface geometry, and electrolyte concentration. It was alsodetermined that the difference in ΔG for the Fc/Fc+ reactionin the DCE and H2O phases, was ∼24 kJ/mol and that therate of the CEPhT reaction is dominated by mass transport.This experimental design, combined with computationalmodeling, provides detailed insights into the mechanisms,

energetics, and kinetics of CEPhT reactions, and could beused to elucidate these details in other chemical systems.

6.5 Imaging buried interfaces

The development of electrochemical microscopy techniquespaves a new way to precisely understand chemical process atsingle-entity level in confined spaces [234]. After threedecades of development, SECM has been shown to bepowerful as a means for measuring numerous surface elec-trochemical properties, but the drawbacks often limit itscapability in a number of applications. For example, theentire sample needs to be immersed in electrolyte, whichmakes it challenging for single-entity measurements. Therecent introduction of scanning electrochemical cell micro-scopy (SECCM) is a significant advancement augmentingthe capabilities of electrochemical microscopy [235]. Incontrast to the solid metal electrodes typically used as theprobe in SECM, the probe in SECCM is a micro-/nanopip-ette filled with electrolyte solution. A cell is formed by themeniscus at the end of the pipette in contact with the samplesurface, which allows for highly localized electrochemicalmeasurements. This technique is very useful for studying iontransfer reactions, phase transformations, and correlatingelectrochemical properties with topographical features, in-cluding localized activity variation on single entitles, such asnanoparticles, nanotubes, and electrocatalysis at grainboundaries [236-238].SECCM is anticipated to help address practical problems

in functioning systems; for instance, through quantifying thekinetics of ion transfer at interfaces, which is an extremelyimportant parameter in energy storage. However, it is diffi-cult to detect the ion transfer and understand the factors in-fluencing ion transfer at buried electrode/electrolyteinterfaces, due to the lack of operando electrochemicalmethods with nanoscale resolution. Recently, a voltammetricSECCM method was employed for the electrochemical in-terrogation of hydrogen evolution reaction (HER) at theburied interface in a model fuel cell cathode. As shown inFigure 21(a), Pt nanoparticles (PtNPs) are deposited onhighly oriented pyrolytic graphite (HOPG) surface andcovered by a Nafion nanofilm as a proton exchange mem-brane; the Nafion-PtNPs interfaces constitutes a buried in-terface. Using SECCM, the localized HER activity ofHOPG, Nafion/HOPG, and PtNPs underneath Nafion can bereadily distinguished via their voltammograms. Owing to thekinetics of HER on Pt being significantly faster than that onthe other sites, it has a highest current enhancement in thevoltammogram (Figure 21(b)). The electrochemical behaviorof the scanned region can also be visualized by an equipo-tential map, which is formed by extracting a single currentfrom the voltammogram at each site. As shown in Figure 21(c), the equipotential map not only shows the current en-

Figure 19 Schematic of the electrochemical cell used to probe CEPhTreactions. A 0.25 mm diameter Pt-Ir wire is placed across the DCE/H2Ointerface, with the DCE phase containing TBAPF6 and Fc and the H2Ophase containing NaCl. The right-side diagram shows the possible me-chanisms of a CEPhT reaction. Reproduced from ref. [233], copyright 2019by the American Chemical Society.

Figure 20 Cyclic voltammetry corresponding to the Pt-Ir wire electrodepositioned at different locations relative to the H2O/DCE interface. (a) Thevoltammetric response with Pt-Ir placed completely in the DCE phase(black), and the Pt-Ir electrode fully emerged in the H2O phase (red). (b)The voltammetric response with the Pt-Ir electrode placed across the H2O/DCE interface. Reproduced from ref. [223], copyright 2019 by theAmerican Chemical Society (color online).

612 Ying et al. Sci China Chem May (2020) Vol.63 No.5

hancement of each site, but also describes the size of buriedPtNPs. SECCM further revealed variation in the HER ac-tivity with changing the thickness of the buried interface.The activity current enhancement rose first and then declinedas the Nafion film thicknesses was increasing from 80 nm to800 nm, and the optimized current enhancement occurred on200 nm Nafion film. This trend is attributed to the balanceamong the ion concentration enrichment, water contentfluctuation, and the restriction for proton transport in Nafion.

7 Perspective

In this review, we have summarized recent developments inthe single entity electrochemistry. The overall motivation ofsingle entity electrochemistry is to learn new chemicalknowledge based on measurements of single molecules,single nanoparticles, single cells and even single tissues.Using nanoscale sensing interfaces, single entity electro-chemistry exhibits high spatial resolution, high temporalresolution and feasible combination with other techniques.Therefore, the single entity electrochemistry is being widelyemployed in biosensing, early disease diagnostics and pre-warning, exploring the mechanisms for energy transforma-tion, revealing the catalysis activities and pathogenic studies.Despite the significant progresses in developing the suitablesensing interfaces and novel sensing mechanisms for singleentity electrochemistry, this field is still in a phase of rapidgrowth and discovery. We end our review here by discussingsome of the scientific challenges to be conquered whileproposing some additional areas for future work.In the field of single molecule detection, the single-bio-

molecule interface provides unprecedented spatiotemporalresolution for single molecule sensing, allowing sufficientobservation time for each analyte and higher throughputrecording mode. However, most of nanopore-based singlemolecule studies are focused on the pure samples and arti-

ficial system instead of applying into the real biosystems.Driven by the motivation for exploring the biologicalcrosstalk, the nanopore-based single molecule tool needsdirectly to acquire electrochemical signal containing singlemolecule information from the single cells. The possiblesolution is to combine the pore-forming biomolecules withnanopipette structure. Such powerful single-molecule sensorcould be used to monitor single-molecule biological pro-cesses in vivo. Moreover, the robustness of these sensorsshould be enhanced to withstand the harsh real samplecondition, and the selectivity should be improved to meet therequirement in simultaneous identification and quantitationof difference species in vivo. Then, the corresponding dataanalysis should be developed to uncover the interaction andkinetics of single molecules in biological crosstalk. Thepossibility of controlling the electrokinetics inside a nano-pipette-biological nanopore hybrid system could be useful tomeet the low detection limit in vivo sensing.For the single nanoparticle detection, the following efforts

are still needed from both activity measurement and structurecharacterization before single nanoparticle electrochemistrycan reach its ultimate goal on the bottom-up SAR. First,intra-nanoparticle features such as facets and defects oftenplay critical roles in regulating the activity of single nano-particles. It thus requires a capability to map the sub-nano-particle-level distribution of electrochemical reactivityinside a single nanoparticle. Many concepts originated fromsuper-resolved fluorescence microscopy, such as super-lo-calization and optical reconstruction, are anticipated to im-prove the spatial resolution of optical electrochemicalimaging [122, 123, 239]. Second, activity measurement hasso far been limited under constant potential (for collision)and potential sweeping conditions (for scanning electro-chemical imaging and optical electrochemical imaging).Despite of the critical roles of impedance measurements intraditional electrochemistry, efforts on single nanoparticleimpedance spectroscopy are still rare [128]. The incorpora-

Figure 21 Visualizing proton reduction on 35-nm radius Pt nanoparticles buried in a 150-nm thick Nafion film. (a) Schematic of SECCM measurement. (b)Voltammetry of HER on HOPG (red), Nafion/HOPG surface (green), and Nafion/PtNP/HOPG (blue). (c) Electrochemical image (121 pixels over a 200 ×200 nm scan area at -1 V vs Ag/AgCl.). The 100 nm-diameter nanopipette contained 25 mM HClO4; ν = 0.1 V/s (color online).

613Ying et al. Sci China Chem May (2020) Vol.63 No.5

tion of temporal (frequency) dimension in impedance spec-troscopy should provide valuable information to decouplemultiple electron transfer processes including electrode-na-noparticle contacts, double-layer charging, ionic migrationand electron hopping within the nanoparticles. From anotheraspect, great efforts are also demanded to improve the cap-ability for in situ characterizing the structural feature ofsingle nanoparticles. Scanning electron microscopes are themost feasible techniques so far to access the size and mor-phology, which, however, often exhibited weak correlationwith the activity due to the lack of internal information suchas crystallinity, atomic arrangement and defects. We ex-pected the adoption of more techniques, such as high re-solution transmission electron microscopy and transmissionX-ray microscopy, to comprehensively characterize the in-ternal structure of single nanoparticles. Energy spectroscopyand optical spectroscopy are anticipated to play more im-portant roles in resolving the chemical states of single na-noparticles. Once the activity and structural of singlenanoparticles can be comprehensively and reliably de-termined, it would be natural for single nanoparticle elec-trochemistry to build a bottom-up methodology to uncoverthe SAR, and to guide the rational design of electroactivenanomaterials with better performance.In single cell analysis, nanoscale devices capable of single-

cell extraction and analysis have thus far been used only inproof of concept studies, this presents a huge arena of bio-logical questions such techniques could be employed to an-swer in the future. The concept of single-cell nanosurgeryrelies on the fact that intracellular contents cannot be mea-sured in situ (i.e., within the intracellular environment) sothat the analyte of interest needs to be first extracted andthen, often after amplification, analysed ex-situ with state ofthe art techniques, like next-generation sequencing of massspectrometry. This is not the case for every intracellularmolecule and with the development of reproducible fabri-cation methods for nanoelectrodes a number of redox activemolecules can be detected and quantified within the cell ofinterest. For example, Ying et al. have reported an elegantsignal amplification approach where only the inner walls of aquartz nanopipette were coated with Au [240]. Upon func-tionalization of the gold layer with catechol the nanoelec-trode showed a remarkable sensitivity to redox-activespecies like NADH. The authors explain the enhanced sen-sitivity due to the generation of H2 nanobubbles at the na-nopipette tip. Remarkably, the authors also showed theability of this nanoelectrode system to detect redox activespecies within a single living cell. Platinised carbon nanoe-lectrodes have been used extensively for the detection ofreactive oxygen and nitrogen species in single cells and thegroups led by Amatore, Mirkin and Ewing amongst othershave demonstrated the potential of this technique for quan-titative intracellular measurement and recently even within

cellular vesicles like lysosomes [241, 242]. Also, there is agreat potential for single molecule manipulation within liv-ing cells as recently demonstrated with nanopore and na-noelectrode-based platforms to enable perturbation andmonitoring of living cells with high spatial and temporalresolution [143, 148, 243]. As thus far nanoscale devicescapable of single-cell extraction and analysis have been usedonly in proof of concept studies, this presents a huge arena ofbiological questions such techniques could be employed toanswer in the future.The unremitting development of electrochemical mea-

surements is expected to address the challenge of preciselydescribing complex electrochemical mechanisms in multiplephases at a confined space. For example, the measurement ofcritical nucleation parameters demands highly sensitive andrapid detection because of the nuclei nanoscopic size and theunstable kinetics. The introduction of nanoelectrodes pro-vides a confined area to precisely study this process with ahigh temporal resolution and repeatability. CEPhT is anotherchallenging scenario happening at all multiple phases inelectrochemistry. Taking advantage of the nanopipette basedelectrochemical approaches, the dimension of the reactionzone is expected to be precisely characterized. The role ofcrystallinity and grain boundaries in influencing electro-chemical ion transfer rates at the buried interface, batterystructure is still largely unexplored, due to the lack of op-erandomethods for probing buried interfaces with nanoscaleresolution. The use of SECCM is expected to advance thestudy of ion transfer reactions and phase transformations atburied interfaces in energy storage with a high spatial re-solution. These measurements will make a significant con-tribution to material, energy, physics as well as chemistry byyielding experimental kinetic rate parameters that test theo-retical predictions.Undoubtedly, the single-entity electrochemistry pushes the

forefront of our ability to make analytical measurements.The further development of advance instrumentation withhigh spatial and temporal resolution, new theory for nanoe-lectrochemistry and new sensing mechanisms, will furtherfacilitate the application of single-entity electrochemistry.

Acknowledgements The authors would like to acknowledge funding fromNational Natural Science Foundation of China (21834001, 21925403,21874070, 21790390, 21790391, 61901171). Y-L. Y. is sponsored by Na-tional Ten Thousand Talent Program for young topnotch talent. P. A. wouldlike to acknowledge funding from the European Union’s Horizon 2020research and innovation programme under the Marie Skłodowska-Curiegrant agreement No 812398. L-Q. M. would like to acknowledge fundingsupported by the Strategic Priority Research Program of Chinese Academyof Sciences (XDB30000000), the National Basic Research Program ofChina (2018YFE0200800, 2018YFA0703501 and 2016YFA0200104), andthe Chinese Academy of Sciences (QYZDJSSW- SLH030). H. W. wouldlike to acknowledgment funding from Office of Naval Research (N00014-19-1-2331), the US Air Force Office of Scientific Research MURI (FA9550-14-1-0003) and the Nanostructures for Electrical Energy Storage (NEES),

614 Ying et al. Sci China Chem May (2020) Vol.63 No.5

an Energy Frontier Research Center funded by the US Department of En-ergy, Office of Science, and Basic Energy Sciences under Award numberDESC0001160. The images reported in Figure 21(b) were acquired usinginstrumentation purchased with support from the Office of Naval ResearchDURIP program (N00014-18-1-2235).

Conflict of interest The authors declare that they have no conflict ofinterest.

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