Boaz Vilozny

Compartmental Genomics in Living Cells Revealed by Single-Cell Nanobiopsy

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.

Reversible cation response with a protein-modified nanopipette.

The calcium ion response of a quartz nanopipette was enhanced by immobilization of calmodulin to the nanopore surface. Binding to the analyte is rapidly reversible in neutral buffer and requires no change in media or conditions to regenerate the receptor. The signal remained reproducible over numerous measurements. The modified nanopipette was used to measure binding affinity to calcium ions, with a K(d) of 6.3 ± 0.8 × 10(-5) M. This affinity is in good agreement with reported values of the solution-state protein. The behavior of such reversible nanopore-based sensors can be used to study proteins in a confined environment and may lead to new devices for continuous monitoring.

Voltage controlled nano-injection system for single-cell surgery

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.

Voltage-Controlled Metal Binding on Polyelectrolyte-Functionalized Nanopores

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.

Reversible thrombin detection by aptamer functionalized STING sensors

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.

Multiwell plates loaded with fluorescent hydrogel sensors for measuring pH and glucose concentration

Fluorescent hydrogels were polymerized directly in multi-well plates at ambient temperature and in the presence of air, producing sensors for measuring pH and glucose concentration. The plates were rapidly analyzed using a fluorescence plate reader. Multiwell pH sensors with good reproducibility among different wells and a dynamic range from pH 6 to 9 were prepared by incorporating a polymerizable pH sensitive fluorophore in the hydrogel. Non-enzymatic glucose sensors comprising a boronic acid-appended fluorescence quencher together with an aminopyrene fluorophore were prepared in a matter of hours in multiwell plates. The sensors showed good reproducibility in response to solutions of glucose at physiological pH. Dried glucose sensors rehydrated with analyte solution performed similarly to freshly prepared hydrogels. The loaded plates are designed for use in high throughput screening applications. Plates were prepared using the redox initiator system metabisulfite/persulfate/iron(II) to generate hydrogels of N,N-dimethylacrylamide crosslinked with N,N′-methylenebisacrylamidein situ.

Enzyme assays with boronic acid appended bipyridinium salts.

In-vitro fluorescent enzyme assays have been developed for sucrose phosphorylase (SPO) and phosphoglucomutase (PGM). These assays make use of a selective carbohydrate sensing system that detects the unlabeled enzymatic products fructose and glucose-6-phosphate. The system comprises 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt as the reporter unit and boronic acid appended viologens as selective receptors with working ranges from 70 microM to 1.0 mM for fructose (SPO) and 190 microM to 2.0 mM for glucose-6-phosphate (PGM). The change in fluorescence can be converted into product concentration, allowing initial reaction velocities and Michaelis-Menten kinetics to be calculated. The assays are also carried out in multiwell plate formats, making them suitable for high-throughput screening of enzyme inhibitors. Rapid PGM inhibition screening is demonstrated with EDTA and LiCl. The PGM assay can also be used for enzyme quantification with a detection limit of 50 ng mL(-1).

Dynamic control of nanoprecipitation in a nanopipette.

Studying the earliest stages of precipitation at the nanoscale is technically challenging but quite valuable as such phenomena reflect important processes such as crystallization and biomineralization. Using a quartz nanopipette as a nanoreactor, we induced precipitation of an insoluble salt to generate oscillating current blockades. The reversible process can be used to measure both kinetics of precipitation and relative size of the resulting nanoparticles. Counter ions for the highly water-insoluble salt zinc phosphate were separated by the pore of a nanopipette and a potential applied to cause ion migration to the interface. By analyzing the kinetics of pore blockage, two distinct mechanisms were identified: a slower process due to precipitation from solution, and a faster process attributed to voltage-driven migration of a trapped precipitate. We discuss the potential of these techniques in studying precipitation dynamics, trapping particles within a nanoreactor, and electrical sensors based on nanoprecipitation.

Copper sensing with a prion protein modified nanopipette

Protein-metal interactions determine and regulate many biological functions. Nanopipettes functionalized with peptide moieties can be used as sensors for metal ions in solution.

Immunoassays Using Artificial Nanopores

Artificial nanopores can be loosely defined as materials possessing one or more nanometersize pores (1-100 nm in diameter). This recent class of nanostructures is generating great interest in the scientific community as a platform for biomolecular analysis. The first fabrication of an artificial nanopore with true nanometer control dates to 2001 (Li et al., 2001) but the application of nanopores for biological studies started 10 years earlier. Following a rapid review of the highlights of 20 years of nanopore science, we explore the advancement of nanofabrication techniques that allowed the creation of individual nanopores, nanopore arrays, and nanoporous materials. Although most of the methods currently used to fabricate nanopores require expensive equipment and highly-skilled technicians, we focus here upon those technologies that allow the fabrication of nanopores at the bench and discuss signal transduction mechanisms that allow nanopores to be used as biosensors. In particular, we review the creative application of nanopipettes, artificial nanopores that can be easily fabricated from inexpensive glass capillaries, as a biosensing platform and discuss their potential for immunosensing.