Nanopipettes—The past and the present

The use of narrow glass tubes in biology has been reported since the discovery of capillary action in the 16th century. In 1904,¹ Albert Barber described a method to fabricate glass capillaries with micrometer-sized pore diameter to capture a single bacterium, and the fabrication of glass pipettes with consistent pore diameters greater than 0.5 µm was established by Ling and Gerard in 1949.² These glass pipettes with definitive pore diameters have been extensively used to study cell physiology.³ One of the earliest applications describing the use of micropipettes for measurement within intracellular spaces was presented by Brown and Flaming in 1980.⁴ Advances in technology and fabrication processes together with driving research in the field of electrophysiology led to the use of pipettes for microinjection and patch clamp applications. Currently, glass pipettes with pore diameters ranging from the micro- to nanoscale are being extensively used in a wide variety of research including analytical chemistry, molecular and cell biology, and sensor development.

The term nanopipettes is generally used to describe glass/quartz pipettes with pore diameters less than 200 nm and having a needle like geometry. The physical properties of nanopipettes make them highly suitable for a wide array of applications including measurement of biomolecules, nanoinjection, and nanobiopsy at subcellular levels as well as electrochemical imaging of cell surfaces. The phenomena of nonlinear current rectification⁵ and steady state voltammetric measurements of ion transfer (IT) kinetics⁶ in nanopipettes have been extensively utilized in several areas of research including cellular and molecular biology and biochemistry since first reported in 1997. Their relatively simple, inexpensive, and accurate means of fabrication has played a pivotal role in the vast adaption of this simple yet powerful tool. In this review, we will discuss in detail the fabrication method employed for the development of nanopipettes, along with the electrochemical characterization of the pipette. We will also examine the use of nanopipettes as sensing probes, monitoring probes for single cells, and scanning probe microscopes (SPMs) by different groups around the globe.

The most commonly used method for the fabrication of nanopipettes utilizes quartz or borosilicate glass capillaries as the precursor. These capillaries are converted into nanopipettes using a laser pipette puller, which is capable of sequential heating and pulling to create two identical nanopipettes. A good example of the laser pipette puller is the model P2000 manufactured by Sutter Instruments, CA, USA. The choice of using borosilicate vs quartz is largely defined by the diameter of the pore required for the specified experiment. Though borosilicate can be easily manipulated, the soft nature of the glass makes it highly difficult to fabricate nanopipettes with diameters less than 80 nm.⁷ Quartz, on the other hand, is more rigid and can be used to manufacture pipettes of varying diameter including ultra-small pipettes with pore diameters as small as 10 nm. Moreover, the low dissipation factor of ∼10⁻⁴ and a dielectric constant of 3.8 of quartz help achieve a low signal to noise ratio in comparison to other glass substrates.⁸ Thus, quartz, with its high mechanical strength⁹ and low electrical noise,⁸ is well suited for both intracellular single cell and electrochemical applications. Another important specification to consider when choosing a capillary is the ratio of the outer diameter (OD) to the inner diameter (ID). This ratio determines the radius at the tip of the nanopipette orifice. The physical properties of nanopipette can be controlled by adjusting five parameters on the P2000 laser puller, namely, HEAT, FILAMENT, VELOCITY, DELAY, and PULL.

• The HEAT parameter defines the power of laser. Output power with a larger heat value corresponding to higher heating temperature.

• FILAMENT specifies the scanning pattern of the laser beam.

• VELOCITY controls the force with which the puller bar travels.

• DELAY governs the time between start of the hard pull relative to the deactivation of the laser.

• PULL determines the force of the hard pull.

A schematic representation of the various steps involved while using the laser pipette puller is shown in Fig. 1. By varying the five parameters mentioned above, nanopipettes with varying orifice diameters and taper lengths can be fabricated. The capillaries need to be thoroughly cleaned using either piranha solution or ethanol prior to use. As an ambient temperature and humidity can affect the heating of the capillary by the laser, a standardized set of parameters may not provide nanopipettes with the same characteristics over different run cycles. Hence, utmost care needs to be exercised while using the laser pipette puller. Representative parameters used for fabricating nanopipettes of different pore diameters at the Pourmand Research Group (University of California, Santa Cruz) are given in Table I. The precursor material used for the fabrication are quartz capillaries having an inner diameter (ID) of 0.70 mm and an outer diameter (OD) of 1.0 mm.

Researchers around the globe have demonstrated the use of alternate techniques for the fabrication of nanopipette. Gao et al.¹⁰ developed a technique wherein the tip of the quartz micropipette was fused, followed by external etching to create a nanopore, while Zhang et al. fabricated nanopipettes with a thicker shank region utilizing a pre-pulled silica nanochannel inside a micropipette.¹¹ The thickness of the shank enabled them to achieve pore diameters from as low as 5 nm to 100 nm. Effort has also been spent toward the development of carbon based nanopipettes. Through the use of catalytic carbon vapor deposition, Kim et al.¹² were able to successfully deposit multiple layers of carbon on the inside and outside of aluminosilicate nanopipettes. Similarly, carbon nanopipettes with pore diameters ranging from 10 nm to 200 nm were also fabricated using noncatalytic CVD by Singhal and co-workers.¹³ The evolution of technology over the last two decades has resulted in the development of inexpensive and simple procedures for the fabrication of nanopipettes.

Morphological characterization of the nanopipette can be achieved using either a transmission electron microscope (TEM) or a scanning electron microscope (SEM). A graphical illustration of the nanopipette is shown in Fig. 2(a), and the SEM image is shown in Figs. 2(b) and 2(c). A typical morphology of the nanopipette consists of the tip, a shank, shoulder, and stem. Of these, the diameter of the tip and the angle of the shank play an important role in determining the resistance of the nanopipette. The relationship between the radii of the tip and of the stem of the pipette and the angle of the cone can be elucidated from the resistance of the pipette using the following equation:

where ρ is the resistivity, l is the length of the shank, r_s and r_t are the radii of the shank and the tip opening, respectively, and θ is the cone angle.

For electrochemical characterization, the nanopipette is filled with a suitable electrolyte and an Ag/AgCl wire electrode is inserted into the stem of the pipette. The nanopipette is then immersed into a bulk solution that contains the reference/counter electrode. Current–voltage changes can then be observed by sweeping the potential between the two electrodes with the help of a potentiostat. A representative image of a typical nanopipette setup is shown in Fig. 3. The electrochemical behavior observed while using a nanopipette is different from that observed using a conventional set up, due to the presence of a diffused double layer (DDL) between the nanopipette and the electrolytic solution.¹⁴ Wei et al.⁵ have documented the role of the DDL in the nonlinear current–voltage characteristics of the nanopipette using different concentrations of KCl and pH. Moreover, they have also observed that rectification occurs only when the diameter of the tip of the nanopipette is comparable to the thickness of the diffuse double layer, with higher asymmetric features witnessed while using higher dilute electrolytes. The effect of electrolyte concentration, pH, and applied potential on current rectification has also been demonstrated by Umehara et al.¹⁵ From both these studies, it is evident that with an increase in the electrolyte concentration, a reduction in the DDL is observed. These experiments indicate the importance of choosing the right electrolyte concentration and pH while using the nanopipette technology.

Functionalization of the inner surface of the nanopipette will alter the current rectification properties. This has been extensively studied by our group.¹⁵ Changes in current rectification can be easily observed by using electrochemical techniques that involve symmetrical wave forms such as sine wave or linear sweeps, while a constant potential will help discriminate the binding events of neutral or slightly charged molecule.¹⁶ 

Over the years, nanopipettes have been extensively employed for the development of sensors for biomedical applications, molecular and cellular biology, as dispensers and aspirators of ultra-small volumes and as probes for a variety of microscopy techniques. A comprehensive review of the recent uses of nanopipettes in these research fields is presented below.

Nanopipettes have been extensively used for the development of biosensors, as they offer the combined advantage of nanoscale dimensions with the selectivity and sensitivity of conventional solid-state biosensors. One of the simplest methods of sensing using nanopipettes relies on the observation of changes in ion current rectification (ICR) when molecules translocate through the pore.^5,14 Over the years, the selectivity and sensitivity of the nanopipette based sensor have been drastically improved by modifying the inner surface of the pipette. Such modifications influence the ion conduction rectification through simple chemical specific changes¹⁵ or through the use of chemically selective layers.¹⁷ More complex modifications include the use of proteins,¹⁷ aptamers,^18,19 and nanoparticles.²⁰ To enable the detection of redox molecules, nanopipettes have been modified with conducting materials including carbon¹¹ and metals.²¹ 

One of the earliest observations in changes to rectification through induced surface chemistry was made by Umehara et al.¹⁵ They studied the use of the cationic polymer poly-l-lysine (PLL) in the nanopipette for biosensing and were able to establish that the rectification observed from the pipettes coated with PLL was amplified and reversed in comparison to the non-coated PLL ones. Umehara et al.¹⁷ have also developed a label-free biosensing platform for protein assays using functionalized nanopipettes. Through their effort, they have demonstrated charge-based biosensing through current rectification observed by the binding of poly-l-lysine (PLL) and polyacrylic acid (PAA) to the quartz nanopipette. In the same body of work, they also established an affinity-based biosensing strategy through the use of biotin-BSA and covalently immobilized IgG. For the biotin-BSA protein assay, a nanopipette functionalized with PLL/PAA was incubated in biotinylated BSA. The addition of streptavidin, which has a high affinity for biotin, resulted in current rectification changes that were not observed in the non-functionalized pipettes. Similarly, current rectifications were also observed in the presence of interleukin-10 and vascular endothelial growth factor (VGEF) using nanopipettes modified with covalently immobilized IgG (Fig. 4).

A label-free technology for thrombin detection was developed by Actis et al.¹⁹ using Signal Transduction by Ion Nano Gating (STING). An aptamer specific for thrombin was immobilized on the pipette surface using PLL. The sensor exhibited selective and reversible thrombin detection both in pure buffer solution and in diluted serum. The development of ion-current-rectification (ICR)-based nanopipette for pH sensing has been well documented. Liu et al.²² developed a cysteine-functionalized nanopipette to produce pH modulated current rectification. For this purpose, researchers developed a facile method for achieving thiol-based linkage on glass nanopipettes, thereby eliminating the need for a gold substrate. The principle used here is based on the reaction between the thiol group in creatine with the maleimide group in N-(2-ami-noethyl) maleimide trifluoro acetic acid via the thiol–Michael addition reaction. A pH range of 2.24–8.00 was successfully measured using the cysteine modified nanopipette. The same group also developed another nanopipette-based pH sensor by adjusting the ratio of 3-aminopropyldimethylethoxysilane (APTES) and (3-triethoxysilylpropyl) succinic acid anhydride (TESP-SA) used in salinization.²³ The relationship between rectification ratio and pH suggested good linearity in the pH range of 3–8. The label-free detection of alkaline phosphatase (ALP) using O-phospho-l-tyrosine (p-Tyr) nanopipettes has also been established.²⁴ During the catalysis of ALP on the nanopipette, the phosphate group of the p-Tyr is removed, resulting in the reduction of negative charge, which is measured as a change in ionic current. Researchers have also used nanopipettes to monitor single molecule immunoreaction.²⁵ This was achieved through the use of a bare nanopipette with a pore diameter of 30 nm and studying the discrimination characteristics of the blockade current, which is characteristic for an individual unlabeled protein. As a proof of concept, the immunoreaction between a single α-fetal protein (AFP) and its specific antibody was detected in an aqueous medium.²⁵ 

A major limiting factor for single molecular detection using nanopipettes is its dependency on diffusion. To overcome this drawback, Freedman and co-workers²⁶ developed a dielectrophoretic (DEP) trap that helped increase the detection limit by 1000-fold. As a proof of concept, they demonstrated the detection of DNA concentration as low as 5 fM at a detection rate of 315 events per minute. Moreover, the incorporation of DEP enabled them to precisely control the volume of molecules that were drawn into the pore along with an increase in detection per unit time. Similarly, a 1000-fold increase in the detection of β-galactosidase and a sixfold increase in the peak amplitude for plasmid DNA have also been accomplished.²⁷ To achieve this sensitivity, the authors relied upon a simple combination of solid-state nanopores with the concept of crowded conditions without the need for any sophisticated surface modification techniques. Researchers have also used resistive pulse technique to detect the folding state of ds DNA.²⁸ The electrophoretic movement of the ds DNA through a nanocapillary of 45 nm resulted in numerous blockage currents that were attributed to the specific folding state of the translocating dsDNA. Bell and Keyser²⁹ employed a novel method for the multiplexed detection of single molecular proteins by conjugating binding sites onto specific oligonucleotides. By integrating a barcoded DNA library with the nanopipette, they were able to detect four different IgG antibodies using a single solid-state nanopore. Their detection system employed a 3-bit library with an average assignment accuracy of 94%.

Nanopipettes have also been used to detect plastic nanoparticles.³⁰ To achieve this, researchers have successfully incorporated single gold nanopores at the tip of the glass pipette. The high Surface Enhanced Raman Spectroscopic (SERS) activity of the gold nanopore results in a clear plasmonic Raman spectrum when a polystyrene nanoparticle of 20 nm translocates through the tip of the nanopipette. This technique is useful for obtaining both qualitative and quantitative information. SERS based detection of dopamine (DA) using nanopipettes has also been established.³¹ The technique involved the fabrication of the nanopipette with a distinctive nanopore and a gold nanoelectrode at the tip of the nanopipette. The nanopore coupled with the gold nanoelectrode could selectively enrich DA, which was detected with high signal to noise ratio using both SERS and electrochemical techniques. A further increase in Raman intensity during SERS was achieved through the use of silver nanoparticles. Cavity based carbon-nanopipette electrodes (CNPE) with pore diameters in the range of 200 nm–400 nm have also been employed for the detection of DA.³² Analytical trapping and enhanced electrical field properties of CNPEs helped achieve the desirable electrochemical properties required for DA detection.

The detection of metal and metal nanoparticles is yet another application for nanopipettes. The use of resistive-pulse for detecting silver nanoparticles as small as 40 nm translocating through the pore of the nanopipette was established by Han et al.³³ The technique relies on the principle wherein the translocation of a conductive nanoparticle through the orifice of the nanopipette creates a high resistance to ionic flow, which can be observed as a sharp drop in the current. Once the nanoparticle leaves the tip of the pipette, the ionic current returns back to the baseline. Actis et al.³⁴ developed a nanopipette to selectively detect copper using the principle of molecular gating. In this study, the concentration-dependent detection of copper was achieved by functionalizing the nanopipette with chitosan and PAA, which enabled the reversible binding of metal ions. The detection of copper using a prion protein (PrP)-modified nanopipette was also established by our group.³⁵ The detection of H₂ and Ag⁺ at a single molecule/ion level was achieved by monitoring the enhanced ionic signatures using a silver coated wireless nanoelectrode (WNE). At a bias potential of −800 mV, the WNE was able to successfully detect 14 H₂ and 24 Ag⁺ molecules from a single signal spike. The reversible binding of Cobalt on nanopipettes functionalized with imidazole-terminated silanes was reported by Sa et al.³⁶ The release of absorbed cobalt ions was achieved by cycling the nanopipettes in solutions of different pH. Apart from playing the role of a sensing device, the reversible binding of the target analyte to the nanopipette also helps in elucidating the kinetic and thermodynamic properties of receptor–target binding.

The nanoscopic pore diameter of nanopipettes has enabled their extensive use for monitoring chemical/biochemical changes within a single cell, as well as inject/aspirate at intracellular levels, as the nanopipettes can enter and exit a single cell without causing damage. Elucidating the molecular biology of single living cells in heterogeneous cell populations helps us to assess and explain the changes in cellular functions within tissues. This is most evident in the case of malignant tumors, which are heterogeneous and can include cells at different stages of transformation.³⁷ Single-cell analysis will help pave the way for a better understanding of biochemical/molecular mechanisms and pathways, aid in the development of therapeutic drugs, and contribute to diagnostics for at-risk individuals.

All cells require glucose for survival, but elevated levels can be indicative of diseases such as cancer, wherein the metabolic pathways of the cell is shifted from mitochondrial oxidative phosphorylation to anaerobic glycolysis that requires excess amounts of glucose.³⁸ Hence, intracellular detection of glucose can help identify abnormal cells in a heterogenic population. Nascimento et al.³⁹ developed a nanopipette based sensor that could detect intracellular glucose levels. To achieve real time monitoring of glucose, the nanopipettes were modified with glucose oxidase (GoX). They observed that the metastatic breast cancer lines MDA-MB-231 and MCF7 expressed higher glucose levels than nonmalignant cells. The nanopipette sensor produced a linear relationship between intracellular glucose levels (0.1 mM–8 mM) and the observed potential response.

As cancer cells exhibit persistently high levels of reactive oxygen species (ROS), intracellular measurement of the superoxide species will benefit studies on tumor progression. Hu et al.⁴⁰ were able to take electrochemical measurements of reactive oxygen and nitrogen species inside living macrophages. They were able to measure H₂O₂, ONOO⁻, NO^•, and NO₂⁻ using platinized carbon nanoelectrodes. Moreover, they employed four different oxidation potentials to obtain the time-dependent concentrations of the different reactive species. Similarly, Ozel et al.⁴¹ developed a ROS sensor for in vitro analysis by modifying the nanopipette with cytochrome C. The principle behind this sensor is that, in the presence of a superoxide molecule O2^*₋, the heme moiety present in cytochrome C will get reduced from Fe³⁺ to Fe²⁺, while O2^*₋ gets oxidized to O₂, resulting in current rectification. The sensor was highly selective toward O2^*₋ and did not respond to the presence of Mg²⁺, Cu²⁺, Zn²⁺, Ca²⁺, Fe³⁺, uric acid, glucose, or ascorbic acid (Fig. 5).

In order to directly observe the activity of β-glucosidase from a single lysosome, Pan et al.⁴² employed a platinum-coated capillary of ∼130 nm filled with β-d-glucopyranoside, glucose oxidase, and 1% Triton X-100. Once the single lysosome came in contact with the Triton X-100 in the nanopipette, β-glucosidase is released, thereby hydrolyzing β-d-glucopyranoside to generate glucose. This glucose is then oxidized by glucose oxidase to produce H₂O₂, which electrochemically oxidizes the platinum ring that produces the electrical signal. The activity of the β-glucosidase in a single lysosome is observed by measuring the H₂O₂ over a period of time. These and other studies at the single cell level provide insights into the localization, expression level, and reaction rates (e.g., activity) of proteins at the subcellular level^43,44 that will help us understand the factors involved in optimal activity levels for intracellular proteins. The intracellular levels of adenosine 5′ triphosphate (ATP) are highly crucial for maintaining physiological and pathological processes. In a related work, the real time intracellular measurement of ATP with good selectivity and high spatiotemporal resolution was successfully established by Zhang et al.⁴⁵ using a polyimidazolium brush (PimB)-modified nanopipette. The selectivity of the assay is attributed to the specific supramolecular interaction between polyimidazolium and ATP coupled with the tunable property of the ion transport within the nanopipette. The basal level and real time monitoring of intracellular ATP was studied using chromaffin cells from wild-type mice and DJ-1 knockout mice.

Though traditional cell biology studies have concentrated on cell population dynamics, elucidating the response of single cells is essential, as it helps us to differentiate cells within a population based on their individual response to a drug/chemical/stimulus.⁴⁶ This can be achieved by developing tools that can specifically introduce material and monitor single living cells while avoiding damage. Toward this end, Laforge et al.⁴⁷ developed an electrochemical nanopipette technique wherein the delivery of attoliter to picoliter volumes of either aqueous or nonaqueous solution was achieved by applying a constant voltage of +600 mV. For this purpose, the nanopipette was filled with an organic solution and immersed in an aqueous solution. On the other hand, Rodolfa et al.⁴⁸ utilized double-barrel nanopipettes for controlled submicron deposition of biomolecules. Our group pioneered the development of a fully automated system with proprietary software that enables the detection of a single cell and injects femtoliter volumes without the need for human intervention.⁴⁹ The simultaneous cell surface detection and precise control of the cargo volume make this technology unique and highly useful. To identify a single cell from a population, a scanning ion conductance microscope was used while a voltage bias between the two barrels of the nanopipette aided delivery. The use of a double barrel nanopipette eliminated the need for an external reference and at the same time enabled the delivery of two different materials. As shown in Fig. 6, the system followed five steps: approach, feedback, penetration, injection, and retraction. Cell viability was monitored by injecting carboxy-fluorescein succinimidyl ester and observing the cell morphology over a period of 27 h. Normal cell division and morphology of daughter cells were observed. Ivanov et al.⁵⁰ developed a nanopipette that combined the controllable delivery of single DNA molecules with simultaneous label-free detection. A major advantage of the developed technology is its efficiency in delivering picomolar concentrations of highly diluted unamplified molecular populations with a precise control over the time of delivery of the first molecule and the total number of delivered molecules through the use of asymmetric voltage pulses. Although the transportation of DNA through the nanopipette under pulsed potentials is attributed to the combination of electrophoretic (EP) and electroosmotic (EO) forces, in this specific translocation, EP was observed to be the dominating force.

Similar to nanoinjection, continuous sampling of intracellular content from individual cells can be achieved through the use of nanobiopsy platform. This is achieved by applying a suitable potential to a nanopipette that is filled with an organic solution and immersed in an aqueous solution.⁵¹ Under these conditions, the application of a negative potential resulted in the movement of the solution toward the lumen of the nanopipette, whereas a positive potential moved the solution toward the outside of the nanopipette. The nanopipette technology can also be employed to aspirate cell contents from the same single cell multiple times during its lifetime. RNA molecules for cDNA synthesis and qPCR analysis have been repetitively isolated from a single cell. Actis et al.⁵² developed a minimally invasive nanosurgical technology to remove minute amounts of total RNA and mitochondrial DNA from a living cell. The application of a constant potential of 500 mV for 5 s caused the aspiration of cell cytoplasm into the nanopipette, while switching the potential to 100 mV stopped the influx. The amount of aspirated material was estimated to be around 50 fl. The analysis of the isolated nucleic acid was carried out using the next-generation sequencing (NGS).

By combining the SICM and the electrochemical syringe, Nashimoto’s research group was able to successfully isolate mRNA and also achieve device automation in the ZYX axes.⁵³ The use of nanostraws for the selective delivery of biomolecules into individual cells has also been documented.⁵⁴ This involved the use of aluminum nanostraws that formed an array of hollow nanowires attached to a microfluidic channel. A combination of diffusion and enhanced electrophoresis resulted in excellent spatial, temporal, and controlled delivery. The nanostraw has also been used for the extraction of mRNA and HSP27 from human induced pluripotent stem cells.⁵⁵ Moreover, the nanostraw was utilized for repetitive sampling over an extended period of time. Through the use of a double barrel nanopipette, nanoscale tweezers were developed to selectively extract DNA and proteins.⁵⁶ The trapping of the biomolecules was achieved through the use of dielectrophoresis caused by the application of non-uniform electric fields. The extraction of the intracellular material provides a new opportunity to understand the underlying mechanisms related to proteomics and genomics.

The use of nanopipettes as probes for scanning probe microscope (SPM) techniques such as scanning electrochemical microscopy (SECM) and SICM has been widely explored. SPM techniques rely on the electrochemical feedback from the redox activity occurring on the surface of the sample immersed in the solution. Employing nanopipettes as probes for electrochemical imaging techniques such as SPM greatly increases the spatial resolution along with the added advantage of improved biocompatibility, small footprint, and simple fabrication process.

Scanning ion conductance microscopy (SICM) uses a nanopipette to scan the surface of the sample without any physical contact, wherein the ion current feedback flowing through the nanopipette controls the distance between the sample and the pipette. A schematic representation of the principle of SICM is shown in Fig. 7. The resolution of the SICM is largely dependent on the pore diameter of the pipette. The principle of SICM was first introduced by Hansma et al.⁵⁷ in 1989. The use of SICM for imaging live cells with a high spatial resolution has been well documented.^57–59 Gorelik et al.⁶⁰ developed a SICM that provided high-resolution images of living cells. The resolution was comparable to that obtained from scanning electron microscopy (SEM) and atomic force microscopy (AFM). Coupling the SICM with a patch clamp enabled them to visualize ion channels in single cells together with anatomic and functional characterization. The same group also used SICM to study the mechanism behind the integrity of the epithelial monolayer of A6 cells.⁶¹ These experiments demonstrate the use of SICM to study biological membranes. Similarly, SICM has been used for the measurement of ion current through a porous polymer membrane, by studying the ion current and topographic images that have been recorded simultaneously.⁶² The measurement of cell volume using SICM was investigated by Korchev et al.⁶³ The use of SICM enabled high resolution characterization of cell volumes from 10⁻¹⁹ l to 10⁻⁹ l without affecting cell functionality. In addition to these applications, SICM has also been used for studying the uptake of nanoparticles by cell.^63,64

One of the major disadvantages of SICM is the requirement for perfectly flat samples as the technique is prone to probe-substrate collision. This was overcome by the introduction of a hopping probe SICM, which could be used for imaging even complex 3D structures.⁶⁵ The robustness of the hopping probe technique is achieved through the principle of imaging selected points on the sample surface. Here the nanopipette is only allowed to approach the spots on the surface where the current is at a fixed value (generally 1%) compared to the reference. Once imaging is completed at the point of interest, the nanopipette is withdrawn to a safe distance before moving on to the next spot on the sample surface. The hopping probe SICM is able to provide a resolution of 20 nm while imaging 3D structures. The simultaneous measurement of pH as well as topography mapping has been demonstrated.⁶⁶ To achieve this dual functionality, an iridium oxide coated carbon electrode was incorporated into one of the barrels of the theta quartz pipette that helped measure the pH while the second barrel was filled with the electrolyte and Ag/AgCl electrode to record the topography of the sample. With the help of SICM, the simultaneous imaging of the topography and surface charge of living cells was achieved⁶⁷ using a bias modulation (BM) scheme. BM involves the modulation of potential between the quasi-reference counter electrode (QRCE) in an electrolyte filled nanopipette and the QRCE in bulk solution. One of the most recent advances in SICM involves the simultaneous mapping of temperature and topography using nanopipettes with apertures as small as 6 nm.⁶⁸ The underlying principle employs the measurement of temperature sensitive ion fluxes near the pipette tip. The miniature size of the pipette tip helps achieve high sensitivity that can differentiate temperature changes as small as 30 mK with a 43 µs resolution.

The concept of SECM was introduced by Bard and Fan in 1986.⁶⁹ This technology can provide information regarding the topography and potential distribution of the sample as well as deliver analytical data. In SECM, the electrode scans the region above a cell surface to measure faradic currents that are the result of the oxidation/reduction of electroactive compounds between the tip and the sample surface. This faradic process is jointly governed by the electron transfer kinetic between the tip and the sample and the mass transfer in the solution.⁶⁹ Moreover, the resolution of SECM is highly dependent on the size and the shape of the probe.⁶⁹ Over the years, SECM has been used for a variety of applications, including electrochemical characterization of the sample surface^58,70–72 and studying membrane transport.^73–76 The use of micro- and nanopipettes as probes for SECM has been extensively studied. By utilizing a 17 nm radius nanopipette, Shen and co-workers⁷⁷ developed an SECM that not only delivers high resolution imaging but also provides the structural properties of single nanopores in a tightly packed space of 93 nanopores/μm². Researchers have also developed an SECM that can provide information about heterogeneous electron transfer kinetics as well as reaction imaging.⁷⁸ This was achieved by combining reagent delivery through the nanopipette with the SECM technique that generally involves the study of electron transfer at the conductive substrate and ion transfer across the liquid/liquid interface supported at the nanopipette tip. This system proved to be highly useful for studying cells in vitro as it eliminated the need for redox mediators in bulk solution and also utilized the high spatial resolution of the electron/ion transfer mode. One of the major disadvantages of the SECM is the need for a small separation distance between the probe and the sample to attain a high resolution. The surface roughness of the probe is a limiting factor for achieving a short distance between the sample and the probe. Elsamadisi et al.⁷⁹ developed a method to polish the tip of the nanopipette that enabled SECM measurements to be carried out at the extremely low distance of 1 nm between the pipette tip and the substrate.

A combined technique utilizing both SECM and SICM (SECM/SICM) was developed to capture high resolution images of cell topography as well as to evaluate the permeation properties of electroactive species through cell membranes.⁸⁰ This system involved the use of a hybrid nanoprobe consisting of a gold nanoring electrode with an outer diameter of 550 nm and an inner diameter of 330 nm and a nanopipette with an aperture of 220 nm. The gold nanoring enables electrochemical measurement for the SECM, while the ion current of the SICM is monitored through the pore of the nanopipette. The authors used SECM/SICM to evaluate the electrochemical response of the enzymes horse radish peroxidase and glucose oxidase as well as to image live cells. Similarly, Comstock et al.⁸¹ developed a nanopipette probe with an integrated ultramicroelectrode (UME) for the simultaneous SECM and SICM measurements. Here, the process of atomic layer deposition was utilized for selectively insulating a gold-coated nanopipette, while focused ion milling was used to precisely expose the UME at the tip of the pipette. With a nanopipette pore diameter of 100 nm and an UME radius of 294 nm, the SECM/SICM was able to successfully image surface features as small as 180 nm. The use of SECM/SICM for the 3-dimensional electrochemical and ion concentration mapping has also been demonstrated.⁸² The use of a carbon nanopipettes (CNP) for the electrochemical resistive-pulse sensing of cellular vesicles inside a macrophage as well as for the measurement of oxygen and nitrogen species (ROS/RNS) contained in the vesicles has been documented.⁸³ A combination of SICM and SECM techniques was utilized for this purpose. SICM was employed to carry out resistive-pulse measurements on the inside as well as on the surface of the living cells, while SECM enabled the insertion of the CNP into the cytoplasm. The detection of cellular vesicles, namely, phagosomes, lysosomes, and/or phagolysosomes, inside RAW 264.7 macrophages as well as extracellular vesicles released by metastatic human breast cells (MDA-MB-231) was successfully accomplished. The authors also reported that the oxidation of ROS/RNS on the carbon surface was observed as upward current spikes, while the translocation of the vesicles caused a current blockage.

In a little over 20 years,^5,6 nanopipettes have emerged as an important tool for a wide variety of applications including sensing, single cell probing, and microscopy. The vast and rapid acceptance of this technology among the research fraternity can be attributed, to a large extent, to the simple and inexpensive fabrication process. The development of materials and methods capable of producing nanopipettes with consistent pore diameters and enhanced rigidity will greatly enable expanding the use of this technology. The use of postprocessing techniques for obtaining nanopipettes with a uniform pore size also needs to be investigated.

The choice of functionalization strategy plays a pivotal role as it provides the required electrochemical characteristics to the nanopipettes. We are of the opinion that the profound advancement in materials science and technology in the development of nanomaterials should be taken advantage of to create nanopipettes with enhanced electrical and chemical properties. These improvements would vastly enhance their versatility, especially in the field of bioanalysis and medical technology.

Data sharing is not applicable to this article as no new data were created or analyzed in this study.