Method for Dynamically Detecting Secretions from Single Cells Using a Nanopore.

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype.

Epigenetic memory emerging from integrated transcription bursts.

Some autonomous bacteria coordinate their actions using quorum-sensing (QS) signals to affect gene expression. However, noise in the gene environment can compromise the cellular response. By exercising precise control over a cell's genes and its microenvironment, we have studied the key positive autoregulation element by which the lux QS system integrates noisy signals into an epigenetic memory. We observed transcriptional bursting of the lux receptor in cells stimulated by near-threshold levels of QS ligand. The bursts are integrated over time into an epigenetic memory that confers enhanced sensitivity to the ligand. An emergent property of the system is manifested in pattern formation among phenotypes within a chemical gradient.

Using a nanopore for single molecule detection and single cell transfection.

We assert that it is possible to trap and identify proteins, and even (conceivably) manipulate proteins secreted from a single cell (i.e. the secretome) through transfection via electroporation by exploiting the exquisite control over the electrostatic potential available in a nanopore. These capabilities may be leveraged for single cell analysis and transfection with single molecule resolution, ultimately enabling a careful scrutiny of tissue heterogeneity.

Kinetics and mechanism of single-stranded DNA adsorption onto citrate-stabilized gold nanoparticles in colloidal solution.

A variety of rapid biomolecular assays under development rely on the selective adsorption of single-stranded DNA onto unfunctionalized, negatively charged, citrate-stabilized gold nanoparticles. We investigate the adsorption mechanism with a study of the binding kinetics and find strong evidence for the dominance of hydrophobic effects including linear compensation between the activation energy and the natural log of the Arrhenius prefactor and the correlation of the adsorption rate in the presence of various salts with the Hofmeister series. These results explain the selectivity for single-stranded over double-stranded DNA adsorption and contradict previous work citing an electrostatic DLVO-like mechanism. Our understanding should facilitate improvements to the selective-adsorption-based assays and, more generally, contribute to the understanding of interactions between like-charged species in aqueous solution.

Wiring Together Synthetic Bacterial Consortia to Create a Biological Integrated Circuit.

The promise of adapting biology to information processing will not be realized until engineered gene circuits, operating in different cell populations, can be wired together to express a predictable function. Here, elementary biological integrated circuits (BICs), consisting of two sets of transmitter and receiver gene circuit modules with embedded memory placed in separate cell populations, were meticulously assembled using live cell lithography and wired together by the mass transport of quorum-sensing (QS) signal molecules to form two isolated communication links (comlinks). The comlink dynamics were tested by broadcasting "clock" pulses of inducers into the networks and measuring the responses of functionally linked fluorescent reporters, and then modeled through simulations that realistically captured the protein production and molecular transport. These results show that the comlinks were isolated and each mimicked aspects of the synchronous, sequential networks used in digital computing. The observations about the flow conditions, derived from numerical simulations, and the biofilm architectures that foster or silence cell-to-cell communications have implications for everything from decontamination of drinking water to bacterial virulence.

Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy.

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.

Direct, concurrent measurements of the forces and currents affecting DNA in a nanopore with comparable topography.

We report direct, concurrent measurements of the forces and currents associated with the translocation of a single-stranded DNA molecule tethered to the tip of an atomic force microscope (AFM) cantilever through synthetic pores with topagraphies comparable to the DNA. These measurements were performed to gauge the signal available for sequencing and the electric force required to impel a single molecule through synthetic nanopores ranging from 1.0 to 3.5 nm in diameter in silicon nitride membranes 6-10 nm thick. The measurements revealed that a molecule can slide relatively frictionlessly through a pore, but regular fluctuations are observed intermittently in the force (and the current) every 0.35-0.72 nm, which are attributed to individual nucleotides translating through the nanopore in a turnstile-like motion.

Biological noise abatement: coordinating the responses of autonomous bacteria in a synthetic biofilm to a fluctuating environment using a stochastic bistable switch.

Noise is inherent to single cell behavior. Its origins can be traced to the stochasticity associated with a few copies of genes and low concentrations of protein and ligands. We have studied the mechanisms by which the response of noisy elements can be entrained for biological signal processing. To elicit predictable biological function, we have engineered a gene environment that incorporates a gene regulatory network with the stringently controlled microenvironment found in a synthetic biofilm. The regulatory network leverages the positive feedback found in quorum-sensing regulatory components of the lux operon, which is used to coordinate cellular responses to environmental fluctuations. Accumulation of the Lux receptor in cells, resulting from autoregulation, confers a rapid response and enhanced sensitivity to the quorum-sensing molecule that is retained after cell division as epigenetic memory. The memory of the system channels stochastic noise into a coordinated response among quorum-sensing signal receivers in a synthetic biofilm in which the noise diminishes with repeated exposure to noisy transmitters on the input of a signaling cascade integrated into the same biofilm. Thus, gene expression in the receivers, which are autonomous and do not communicate with each other, is synchronized to fluctuations in the environment.

Direct visualization of single-molecule translocations through synthetic nanopores comparable in size to a molecule.

A nanopore is the ultimate analytical tool. It can be used to detect DNA, RNA, oligonucleotides, and proteins with submolecular sensitivity. This extreme sensitivity is derived from the electric signal associated with the occlusion that develops during the translocation of the analyte across a membrane through a pore immersed in electrolyte. A larger occluded volume results in an improvement in the signal-to-noise ratio, and so the pore geometry should be made comparable to the size of the target molecule. However, the pore geometry also affects the electric field, the charge density, the electro-osmotic flow, the capture volume, and the response time. Seeking an optimal pore geometry, we tracked the molecular motion in three dimensions with high resolution, visualizing with confocal microscopy the fluorescence associated with DNA translocating through nanopores with diameters comparable to the double helix, while simultaneously measuring the pore current. Measurements reveal single molecules translocating across the membrane through the pore commensurate with the observation of a current blockade. To explain the motion of the molecule near the pore, finite-element simulations were employed that account for diffusion, electrophoresis, and the electro-osmotic flow. According to this analysis, detection using a nanopore comparable in diameter to the double helix represents a compromise between sensitivity, capture volume, the minimum detectable concentration, and response time.

High-yield transfer printing of metal-insulator-metal nanodiodes.

Nanoscale metal-insulator-metal (MIM) diodes represent important devices in the fields of electronic circuits, detectors, communication, and energy, as their cutoff frequencies may extend into the "gap" between the electronic microwave range and the optical long-wave infrared regime. In this paper, we present a nanotransfer printing method, which allows the efficient and simultaneous fabrication of large-scale arrays of MIM nanodiode stacks, thus offering the possibility of low-cost mass production. In previous work, we have demonstrated the successful transfer and electrical characterization of macroscopic structures. Here, we demonstrate for the first time the fabrication of several millions of nanoscale diodes with a single transfer-printing step using a temperature-enhanced process. The electrical characterization of individual MIM nanodiodes was performed using a conductive atomic force microscope (AFM) setup. Our analysis shows that the tunneling current is the dominant conduction mechanism, and the electrical measurement data agree well with experimental data on previously fabricated microscale diodes and numerical simulations.