Functionalized nanopipettes: toward label-free, single cell biosensors
- 1.8k Downloads
Nanopipette technology has been proven to be a label-free biosensor capable of identifying DNA and proteins. The nanopipette can include specific recognition elements for analyte discrimination based on size, shape, and charge density. The fully electrical read-out and the ease and low-cost fabrication are unique features that give this technology an enormous potential. Unlike other biosensing platforms, nanopipettes can be precisely manipulated with submicron accuracy and used to study single cell dynamics. This review is focused on creative applications of nanopipette technology for biosensing. We highlight the potential of this technology with a particular attention to integration of this biosensor with single cell manipulation platforms.
KeywordsNanopipette Label-free Single cell detection Single molecule detection Biosensor Nanopore
Increasing demand for more sensitive analytical and diagnostic tools for the identification of biomolecules has benefited from advanced nanofabrication techniques and the significant strides made in the biosensing and biomedical research fields in recent years. New biosensing techniques such as nanostructured optical fiber arrays , carbon nanotube biosensors , nanowires , and nanoparticles  provide improved sensitivity and often require less complicated instrumentation than existing molecular detection technology. A relatively new label-free technology, the nanopipette illustrates the trend toward exciting new approaches for biosensing applications.
A pipette can be loosely defined as a hollow structure in which the cavity acts as a passage for the dispensation of fluid from one region to the next. Typically, pipettes are categorized by their volume—microliter, nanoliter, pico, and zeptoliter pipettes . The volumetric definition generally provides a good guideline on the pipette dimensions. However, when the pipette is used as a biosensor (rather than to dispense a liquid volume), the tip diameter is of greater interest than the overall volume. One can conceivably apply a similar SI standard (micro-, nano-, etc.) to the tip diameter. But a more descriptive subdivision is more desirable and often more informative.
Although the SI definitions of the prefixes are clear (micro-, nano-, etc.), the modifiers to the prefixes are often not bound by the same set of standards. For instance, electrochemists had previously struggled with the nomenclature of electrodes based on their size. It became necessary to have some standardization for the description of the electrodes. Electrodes with a diameter of <200 nm are defined as nanoelectrodes, whereas microelectrodes are normally considered to be ∼0.2–20 µm in diameter, according to the IUPAC classification [6, 7]. Applying similar standardization, one can define nanopipettes to be hollow, free standing structures with an opening in the 1–200 nm range. Although a definition based on simple geometrical parameter is exhaustive, nanopipettes with openings smaller than ∼100 nm show non-uniform electrochemical characteristics stemming from the physical properties at the nanoscale, leading to new physical behavior, as described below in the “Electrochemistry of nanopipettes” section.
While nanopipettes and solid-state nanopores are fundamentally similar in terms of the sensing principle, the fabrication processes and the configuration of the components suggest that the two technologies should be considered two different entities. Here we explore and review the fundamental elements of nanopipettes and recent works from various groups related to the creative use of nanopipettes as biosensors. The applications of nanopipettes to investigate single molecule biophysics and to image cells at the nanoscale [8, 9] have been recently reviewed and will not be discussed here.
Nanopipettes offer a unique biosensing platform that is potentially capable of detecting single molecules near their most sensitive region. Typically an elongated cone, the dimension and geometry of the nanopipette tip orifice are crucial for the development of a new sensitive biosensing platform. The nanopipette has a size comparable to that of DNA and protein. The interaction of such a biomolecules with the nanopipette pore causes two unique events—temporary blockage due to ionic/molecular translocation through the nanopipette and/or permanent blockage resulting from binding to a tip that has been functionalized with specific recognition molecules. Each of these interactions causes a distinctive change of the nanopipette electrical behavior. The electrical changes can then be detected with simple electrochemistry without the need for labeling the molecule of interest. The detection principles are detailed in the latter part of this review.
Our group, along with others, has reported the use of different materials for nanopipette fabrication. The diversity of prospective materials, glass or metallic, makes nanopipettes extremely versatile systems toward biosensor development. Each scheme can be tailored with a specific material for optimal sensitivity and robustness. While routinely used as electrochemical sensors [10, 11], nanopipettes can readily couple to other instrumentation for secondary detection  or parallel verification . The potential combination with other analytical techniques has led to a multitude of chemical and biochemical detection approaches [14, 15, 16]. In this review, we will focus on the recent novel, label-free, biosensing applications using nanopipette. Also, we will explore the potential of similar structures to be used as biosensors and diagnostic tools.
Nanopipette materials and fabrication
Interest in nanopipettes was triggered by significant advances in nanofabrication techniques . A wide range of nanopipettes have been fabricated under different names with various materials for chemical and biochemical sensing. One of the most attractive features of nanopipettes is the simplicity of the fabrication. Due to their inherent stability and unique thermal properties, glass substrates have been a leading choice for nanopipette fabrication. The low melting temperature of borosilicate compared to fused quartz makes the fabrication of sub-100 nm nanopipettes is extremely difficult . Besides this technical limitation, quartz offers superior material properties for a variety of research applications. For example, quartz is stronger than other glasses and can facilitate penetration through tough tissues which would normally break borosilicate pipettes . In electrochemical applications, quartz has the lowest electrical noise among all types of glass available [20, 21]. Furthermore, quartz contains none of the metals used in conventional glasses, making it virtually free from fluorescence when illuminated . As a result, fused quartz is the predominant glass substrate for fabricating nanopipettes .
Significant effort has been invested in fabricating nanopipettes from materials other than glass. Kim and colleagues  fabricated carbon-based nanopipettes with large aspect ratios (length/diameter) based on the glass-pulling technique. In this technique, carbon layers are deposited onto the exterior and interior of the pulled aluminosilicate nanopipettes using catalytic chemical vapor deposition. The exterior carbon layer and the glass layer are subsequently removed by chemical etching, exposing the interior carbon nanopipette tip structure.
Freedman and coworkers  fabricated carbon nanotube-tipped probe using magnetic techniques. In this approach, a magnetized carbon nanotube (mCNT) is affixed to the tip of a conventional glass nanopipette using magnetic manipulation. The resulting mCNT-tipped nanopipettes were sufficiently robust that they could be used to penetrate cell membranes. The nanopipette also demonstrated fluidic transport ability through the opening of the mCNTs.
In general, nanopipettes can be fabricated with many different materials, each providing unique characteristics and electrochemical properties. The one significant advantage of adopting nanopipettes is that the fabrication process is extremely simple, or even considered fab-less, in some cases.
Electrochemistry of nanopipettes
Current rectification has been shown to be affected by electrolyte concentration, pH, and applied voltage . Current rectification is only observed at electrolyte concentrations at or below 100 mM. No significant current rectification was reported at high electrolyte concentrations. Consistent with existing electrochemical literature, the ddl thickness is reduced with an increase of electrolyte concentration, e.g., characteristic ddl thicknesses are about 3, 1, and 0.3 nm in 0.01, 0.1, and 1 M KCl solutions, respectively . At high salt concentration, the ion flow becomes insensible to surface charges. Therefore the overall electrolyte concentration in the system is crucial for nanopipette experiments.
Current rectification can be modulated by functional layers deposited or covalently attached to the nanopipette opening region. Poly-l-lysine, a polypeptide bearing positively charged amino groups, can be physisorbed on the negatively charged nanopipette surface . The protonated amino groups invert the current rectification (Fig. 2c). Similar results were observed with nanopipette modified with cationic dendrimers  and in a protein-binding study using PEG-modified nanopipette-like structures .
The rectification coefficient is a useful parameter for monitoring the variation of nanopipette electrical response with the introduction of a functionalized layer to the nanopipette tips. Uncoated glass nanopipette surfaces (e.g., quartz or borosilicate) induce a negative current rectification (r > 1). Functionalization of the nanopipette quartz surface with positively charged polyelectrolytes, such as poly-l-lysine, inverts the current rectification (r < 1). In general, any charged molecule captured at the nanopipette tip will modify the surface charge density at the nanopipette and the binding can be monitored by plotting the variation of the rectification coefficient over time. Such data provide details of binding or adsorption events that occur at the tip orifice.
Equation 3 gives an approximate relationship between the nanopipette orifice size and by simple resistance measurements and it is in good agreement with radii estimated by the electron microscope [23, 33].
It is important to point out that the confinement of the resistance at the very tip of a nanopipette makes this technology extremely appealing for the development of ultrasensitive biomolecular detection platforms capable of detecting single molecule events [8, 34].
Generally, the input voltage is crucial to the sensing and detection mechanism. Symmetric waveforms, such as sine waves or linear sweep, are useful for investigating the binding of charged molecules (such as DNA) and binding kinetics can be monitored by following the variation of the rectification coefficient over time. On the other hand, a constant voltage will not allow any monitoring of the change in the rectification properties but it is more suitable for discriminating binding events of neutral or slightly charged molecules. In addition, a constant voltage can concentrate molecules at the tip though diffusion, electro-migration, and electro-osmotic flow .
Nanopipettes as electrochemical biosensors
The process of trapping and translocation of these conjugates through the nanopipette is not the result of a simple diffusion mechanism with Gaussian distribution as it would be for Brownian motion, but a mechanism by which molecules must cross an energy barrier in order to enter or escape across the pore. This first attempt to employ a nanopipette as a biosensor clearly showed some potential, and an appropriate surface chemistry will enhance this approach, turning it into a label-free assay for DNA without the use of nanoparticles.
The goal of their work was to prove that biomolecular interactions can be detected in a label-free manner through nanopipette technology. However, improvement in the technology can be made, for example, by limiting the functionalized surface area to prevent the capture of targets on the sidewalls away from the sensing region. Another option is to take advantage of the accumulation of molecules in and around the tip region under constant voltage .
To fully realize the potential of nanopipette as an analytical biosensor, the nanopipette response to analyte concentration should be systematically investigated. Ding et al. proposed an aptamer-encoded nanopipette to detect immunoglobulin E and a ricin, a 64 kDa glycoprotein toxin, and explored the effect of analyte concentration in this system. For the immobilization of aptamer on the nanopore, they followed a method that was successfully established to immobilize proteins on silicon nanowires . The glass surface in the pore was silanized with aldehyde methoxysilane, followed by attaching amino-terminated DNA or RNA aptamers to the aldehyde-terminated glass surface in sodium cyanoborohydride. The electrical measurements were performed in a PBS solution, and the ionic current at the nanopore was monitored as a function of time under a constant applied voltage.
Summary of recent nanopipette biosensing applications
Limit of Detection
Small proteins (IL-10, VEGF)
500 fM (IgE)
>130 min at 500 fM
2.8 nM (Ricin)
DNA labeled with Au nanoparticle
Nanopipettes and electrophysiology
Nanopipettes and micromanipulators can be integrated for single cell imaging and nanoinjection. We will not discuss all the different applications of the scanned nanopipette, as this topic has been recently reviewed by Klenerman and coworkers . However, some interesting features can lead to a unique biosensor platform capable of a sensitive analysis ex vivo down to the single cell level. Hansma and colleagues  first demonstrated the ability of glass nanopipette to image the topography of non-conducting surfaces immersed into electrolyte solutions. A variation in current is observed when the nanopipette tip approaches the surface of the sample. Therefore, the measured ionic current can be used as input to a feedback control loop keeping the distance between the tip and the sample constant during scanning . However, small electrolytic changes in electrodes, e.g., accumulation of charged molecules inside the pipette, will also affect the measured current. To address this issue, a distance-modulated control mechanism has been developed for reliable non-contact imaging over the surface of a live cell .
high cell survival rate
precise control of the amount of material delivered
possibility of a voltage or pressure driven injection
applicability of the platform for the delivery of multiple (bio)molecules
Work from many research groups highlights the advances in the use of nanopipettes for injection. Bruckbauer et al. delivered individual fluorescently labeled probe molecules to the plasma membrane through a nanopipette. Single molecule fluorescence tracking was used to validate the method. They studied the diffusion of individual membrane glycoproteins labeled with a fluorescent dye in different surface domains of boar spermatozoa . Piper et al. demonstrated the local control of sodium-sensitive flagellar motor in single Escherichia coli cells by dosing sodium via a nanopipette . Laforge et al. filled a nanopipette with a water-immiscible organic solvent and immersed it in an aqueous solution . The electrochemical attosyringe takes advantage of the phenomenon that the application of voltage across the liquid/liquid interface changes the surface tension . The resulting force is sufficiently strong to induce the flow of liquid into/out of the pipette. They have successfully used this effect to deliver femtoliters of aqueous solution into mammalian cells in culture. Cell integrity after injection was confirmed by trypan blue-exclusion. Similar experiments were performed with carbon nanopipettes . Vitol et al. introduced a SERS-active carbon nanopipette for intracellular analysis . SERS functionality is added by incorporating gold nanoparticles on the outer surface pipette tip. The technique allows the accurate tracking of the tip location within the cell. SERS spectra obtained with the nanopipette from within the nucleus are clearly different from those obtained within the cytoplasm and contain typical features associated with DNA.
Conclusions and outlook
Recently, many researchers have focused on applications of nanopipettes for single cell penetration to study intracellular compartments, and there is a growing interest in the exploration of functional phenomena directly inside a single (pathogenic) cell. Recent advances combine the sensitivity and the selectivity of nanopipettes as a biosensor with the ease and the precision of manipulation of such sensor into well-defined region of a cell surface or compartment. Although nanopipettes are recent inventions, they are a promising tool for combining single cell analysis and cell manipulation. We believe that the nanopipette will become an essential tool for electrophysiological and medical research in the near future.
This work was supported in part by grants from the National Aeronautics and Space Administration Cooperative Agreements NCC9-165 and NNX08BA47A, National Institutes of Health [P01-HG000205], the National Science Foundation [DBI 0830141].
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- 7.Cunningham A (2000) Introduction to Bioanalytical Sensors. Wiley, NY, p 29.Google Scholar
- 12.Gorelik J, Shevchuk A, Ramalho M, Elliott M, Lei C, Higgins CF, Lab MJ, Klenerman D, Krauzewicz N, Korchev Y (2002) Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: application to single virus-like particle entry into a cell. Proc Natl Acad Sci USA 99:16018–16023CrossRefGoogle Scholar
- 22.Zuazaga C, Steinacker A (1990) Patch-clamp recording of ion channels: interfering effects of patch pipette glass. News Physiol Sci 65:1666–1677Google Scholar
- 29.Bard AJ, Faulkner LR (eds) (1980) Electrochemical methods: fundamentals and applications. Wiley, New YorkGoogle Scholar
- 32.Sakmann ENB (ed) (1995) Single-channel recording. New YorkGoogle Scholar
- 33.Lavallée OSM, Hébert N (ed) (1969) Glass microlectrodes. New YorkGoogle Scholar
- 41.Liming Ying AB, Zhou D, Gorelik J, Shevchuk A, Lab M, Korchev Y, Klenerman D (2005) The scanned nanopipette: a new tool for high resolution bioimaging and controlled deposition of biomolecules. PCCP 7:2859–2866Google Scholar
- 44.Girault H, Schiffrin DJ (eds) (1989) Electroanalytical chemistry. Marcel Dekker, New YorkGoogle Scholar