The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

b S Supporting Information E lectrical devices that can measure ion current through a nanopore are gaining attention as a new way to design sensors with nanoscale resolution. 1,2 Receptors immobilized to nanopore-based ion current sensors have included proteins, 3À5 enzymes, 6 DNA, 7 aptamers, 8 ligands, 9,10 and small biomolecules, 11,12 allowing nanoscale measurement of a variety of analytes. Sensing by modulation of ion current in functionalized nanopores is distinct from the technique of resistive-pulse sensing, which is used to characterize macromolecules by translocation through a pore. 13 To distinguish this mechanism in functionalized nanopipette sensors, 14 we coined the term signal transduction by ion nanogating (STING), evoking both the role of ion current and the needlelike shape of the nanopipette. Essential to the sensitivity of many solid-state nanopore sensors is selective permeability of electrolytes, or ion current rectification, when a bias is applied across the nanopore. Ion current rectification (ICR) arises from the selective interaction between ions in solution and the surface of a charged, asymmetrically shaped nanochannel or conical nanopore. 15 Nanomaterials exhibiting ICR and used as sensors include track-etched nanopores in polymer membranes 16 and quartz nanopipettes. 17 In either case, the surface modification of nanopores with appropriate receptors is a key challenge to sensor development.To date, the reversible binding of analytes with nanopore sensors has proven challenging. However, this is a critical issue if such devices are to be used for applications such as continuous monitoring or repeated measurements with one sensor. Multiple uses for a single sensor will also overcome problems in reproducible nanopore fabrication, which limits quantitative measurements for many sensors reported in the literature. For applications using

Manipulation and analysis of single cells is the next frontier in understanding processes that control the function and fate of cells. Herein we describe a single-cell injection platform based on nanopipettes. The system uses scanning microscopy techniques to detect cell surfaces, and voltage pulses to deliver molecules into individual cells. As a proof of concept, we injected adherent mammalian cells with fluorescent dyes.

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Signal Transduction by Ion NanoGating (STING) is a label-free technology based on functionalized quartz nanopipettes. The nanopipette pore can be decorated with a variety of recognition elements and the molecular interaction is transduced via a simple electrochemical system. A STING sensor can be easily and reproducibly fabricated and tailored at the bench starting from inexpensive quartz capillaries. The analytical application of this new biosensing platform, however, was limited due to the difficult correlation between the measured ionic current and the analyte concentration in solution. Here we show that STING sensors functionalized with aptamers allow the quantitative detection of thrombin. The binding of thrombin generates a signal that can be directly correlated to its concentration in the bulk solution.

Most of the research in the field of nanopore-based platforms is focused on monitoring ion currents and forces as individual molecules translocate through the nanopore. Molecular gating, however, can occur when target analytes interact with receptors appended to the nanopore surface. Here we show that a solid state nanopore functionalized with polyelectrolytes can reversibly bind metal ions, resulting in a reversible, real-time signal that is concentration dependent. Functionalization of the sensor is based on electrostatic interactions, requires no covalent bond formation, and can be monitored in real time. Furthermore, we demonstrate how the applied voltage can be employed to tune the binding properties of the sensor. The sensor has wide-ranging applications and, its simplest incarnation can be used to study binding thermodynamics using purely electrical measurements with no need for labeling.

Nanofluidic structures share many properties with ligand-gated ion channels. However, actuating ion conductance in artificial systems is a challenge. We have designed a system that uses a carbohydrate-responsive polymer to modulate ion conductance in a quartz nanopipette. The cationic polymer, a poly(vinylpyridine) quaternized with benzylboronic acid groups, undergoes a transition from swollen to collapsed upon binding to monosaccharides. As a result, the current rectification in nanopipettes can be reversibly switched depending on the concentration of monosaccharides. Such molecular actuation of nanofluidic conductance may be used in novel sensors and drug delivery systems.