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.

ADVERTISEMENT RETURN TO ISSUEPREVLetter to the EditorNEXTGene Expression in Electron-Beam-Irradiated Bacteria in Reply to "Live Cell Electron Microscopy Is Probably Impossible"Eamonn Kennedy†, Edward M. Nelson†, John Damiano‡, and Gregory Timp*§View Author Information† Department of Electrical Engineering, University of Notre Dame, Notre Dame, Indiana 46556, United States‡ Protochips, Inc., Morrisville, North Carolina 27560, United States§ Departments of Electrical Engineering and Biological Science, University of Notre Dame, Notre Dame, Indiana 46556, United States*E-mail: [email protected]Cite this: ACS Nano 2017, 11, 1, 3–7Publication Date (Web):January 24, 2017Publication History Received30 September 2016Published online24 January 2017Published inissue 24 January 2017https://pubs.acs.org/doi/10.1021/acsnano.6b06616https://doi.org/10.1021/acsnano.6b06616letterACS PublicationsCopyright © 2017 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views1763Altmetric-Citations18LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (1 MB) Get e-AlertscloseSUBJECTS:Assays,Fluorescence,Genetics,Irradiation,Membranes Get e-Alerts

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.

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.

We report direct and simultaneous measurements of the forces and currents associated with the translocation of a single-stranded DNA molecule tethered to an AFM cantilever (Figure a) through synthetic pores 1.2 to 3.5 nm in diameter in 8±2 nm thick silicon nitride membranes. These measurements were performed to determine the force required and the electrical signal available for sequencing a single molecule of DNA in a pore small enough to affect the configuration of the molecule. The measurements revealed that ssDNA either translocated the nanopore in a "stick-slip" motion characterized by multiple stretching and rupture events or slid with a relatively constant net force between 10 and 60 pN (Figure b). While the tip moved at a constant velocity, minute <1 pN and <20 pA fluctuations in the force and current, respectively, were observed every 0.35-0.60 nm (Figure c) in homo/heteropolymers of ssDNA, which were attributed to individual nucleotides translating through the nanopore in a turnstile-like motion. These results indicate that synthetic nanopores <2 nm in diameter may offer the resolution to sequence individual bases of DNA, provided the molecule slides through the pore.

It is now possible to manipulate individual molecules using a nanopore to read DNA and proteins, or write DNA by inserting mini-genes into cells. Furthermore, development of these methodologies will kick open the door to new biology and chemistry that has been logistically intractable previously. Nanopore technology will place molecular and sub-molecular analysis within the reach of the typical bench-top scientist or clinical lab-no longer limited to genomics or mass spectrometry specialists. Moreover, the prospects for synthetic biology-using nanopores to program or reprogram cells-are promising as well, but have been examined only at the level of a single cell, so far.

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.

The ecology in a biofilm—i.e., how the cells relate to each other and their environment—can offer competitive advantages over an autonomous, free-swimming, planktonic environment such as nutrient acquisition via cross-feeding of populations and increased resistance to biocides. To explore this ecology, we investigated the physical parameters governing prokaryotic cell-to-cell signaling in a simple model of a biofilm created using live-cell lithography, comprising bacteria that are genetically engineered to transmit and receive quorum-sensing signals. These experiments, along with the numerical simulations that mirror them, revealed that gene expression resulting from transmitter to receiver communications was vitally dependent on the location within the biofilm elements and the epigenetic memory associated with a bistable switch in the receiver gene, which were both easily accessible in the model. Three-dimensional biofilm models with open channels that include still more complex communication networks for the study of wound repair are in the offing.

The secretome offers the prospect of new biomarkers for the diagnosis of disease and a promising approach to drug discovery. However, these proteins are secreted in only minute amounts. We propose to use a nanopore to both transfect single cells with nucleic acids and detect secreted protein.

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.

Tissue engineering strives to create functional tissue for organ replacement and regenerative medicine by combining living cells with a porous scaffold that promote growth and differentiation. Recent attempts to replace tissue with autologous cells on a biodegradable scaffold have been frustrated by inflammatory and healing responses. We speculate that without vascularization and hierarchical organization, gradients in effector molecules and basic nutrients can develop in engineered tissue due to the competition between convection and diffusion, cell consumption and production that compromises cell function and viability. In particular, once implanted in the patient, the cells in the engineered tissue consume the available oxygen and nutrients within a few hours, while it can take several days for the growth of new blood vessels (angiogenesis) to deliver nutrients to the implant. We have created using "live cell lithography" an in vitro model that emulates the in vivo microenvironment found in human capillaries with micron-scale precision. To create a capillary, we used a microfluidic to convey human umbilical vein endothelial cells (HUVECs) into multiple microarrays of optical tweezers, which are used to precisely position cells in a 3D hydrogel scaffold (see figure).

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.

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.

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.

Biofilms are the archetype for smart materials where microcolonies of live bacteria and layers of dead cells are encapsulated in a hydrated matrix of polysaccharides, proteins and exopolymeric substances. We engineered synthetic biofilms to study their ecology_i.e. the cells' relationship to each other and their environment. In particular, we investigated the physical parameters governing prokayrotic cell-to-cell signaling in a vascularized model of a biofilm, comprised of bacteria that are genetically engineered to transmit and receive so-called quorum-sensing signals based on the lux operon functionally linked to fluorescent reporters. Numerical modeling of these experiments reveals that gene expression is vitally dependent on the location within the biofilm and proximity to microchannels in the array, elements easily accessible in the model. Moreover, we observe synchronization in fluorescence from the microcolonies comprising the biofilm in a repetitive, pulsatile environment as shown in the figure. We find that the concentration of luxR mRNA varies measurably with C6HSL concentration and time indicating autoregulation of luxR. Moreover, flow cytometry data identifies two phenotypes in the cell population: one with a high C6HSL threshold; another with a lower threshold, indicating bi-stability associated with positive autoregulation of luxR.

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.