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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.

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.

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.

The ability to probe an interface beneath a layer of living cells in situ without the need for labeling and fixation has the potential to unlock some of the key questions in cell biology and biointerfacial phenomena. Here, we show that vibrational sum frequency generation (SFG) spectroscopy can be used to detect alkanethiol self-assembled monolayers (SAMs) buried underneath a layer of living erythrocytes (ECs). SFG spectra with and without ECs showed the spectral signatures typical of these SAMs, indicating that the signal was being generated solely by the SAM and was not influenced by the presence of cells. Direct comparison of infrared spectroscopy to SFG measurements of cells adhered on a fibronectin layer showed that the SFG signal emanated solely from this layer. These results have important implications for the characterization of surfaces in biomedical, environmental, and industrial applications.

The thermo-responsive behaviour of thiol modified poly(N-isopropylacrylamide) (pNIPAM) films immobilized on gold are probed by in situ broadband sum-frequency generation (SFG) spectroscopy. The pNIPAM films were prepared by atom transfer radical polymerization (ATRP) using a nitro-biphenyl-thiol (NBT)-SAM on a polycrystalline gold surface as a substrate. Additionally, Raman and infrared reflection absorption spectroscopy (IRRAS) are applied to spin-coated pNIPAM films. Molecular groups involved in the reorientation and disordering of the polymer chains during the LCST (lower critical solution temperature) transition of pNIPAM are identified. The characteristic vibrations of the CH(3) groups show a gradual reorientation of the isopropyl groups within the pNIPAM film and instantaneous reorientation of the outermost CH(3) groups around 32 degrees C.

Capillaries pervade human physiology. The mean intercapillary distance is only about 100 μm in human tissue, which indicates the extent of nutrient diffusion. In engineered tissue the lack of capillaries, along with the associated perfusion, is problematic because it leads to hypoxic stress and necrosis. However, a capillary is not easy to engineer due to its complex cytoarchitecture. Here, it is shown that it is possible to create in vitro, in about 30 min, a tubular microenvironment with an elastic modulus and porosity consistent with human tissue that functionally mimicks a bona fide capillary using “live cell lithography”(LCL) to control the type and position of cells on a composite hydrogel scaffold. Furthermore, it is established that these constructs support the forces associated with blood flow and produce nutrient gradients similar to those measured in vivo. With LCL, capillaries can be constructed with single cell precision—no other method for tissue engineering offers such precision. Since the time required for assembly scales with the number of cells, this method is likely to be adapted first to create minimal functional units of human tissue that constitute organs, consisting of a heterogeneous population of 100–1000 cells, organized hierarchically to express a predictable function.

The secretome of a single living cell contains the totality of its secreted proteins,(1) and therefore can act as a fingerprint by which to identify cell type. Although between 10 % and 20 % of the human genome encodes proteins that are secreted, measuring the secretome from an individual living cell is challenging as the secreted proteins are present in vanishingly small concentrations due to the very large dilutions involved.However, a nanopore is able to detect single proteins through the distinctive blockage profile that develops in the ionic conductance current when a protein passes through the nanopore.(2,3) Using a synthetic nanopore in a silicon membrane we investigate the distinctive blockage patterns, in essence the fingerprint, that arise from a single living cell. The cell is placed in proximity to the nanopore using optical tweezers and held stationary. The ionic conductance current is measured across the nanopore, and translocation events (distinct blockage currents) are observed and measured. When the events are plotted in scatter plots (as dwell time versus average blockage current) the distinct fingerprint of individual cells can be observed. For instance, lymphoma cells (U937) and breast cancer cells (MCF7) produce distinct event patterns that enable them to be distinguished. This shows for the first time cell identification based entirely on the secretome, measured using a simple, non-invasive, non-destructive nanopore.(1) Skalnikova, H.; Motlik, J.; Gadher, S.; Kovarova, H. Proteomics, 2011, 11 691-708.(2) Nelson, E. M.; Kurz, V.; Shim, J.; Timp, W.; Timp, G. Analyst, 2012, 137, 3020.(3) Kurz, V.; Nelson, E. M.; Shim, J.; Timp G. ACS Nano, 2013, 7(5), 4057-4069

We report the development of a single cell gene delivery system based on electroporation using a synthetic nanopore, that is not only highly specific and very efficient but also transfects with single molecule resolution at low voltage (1 V) with minimal perturbation to the cell. Such a system can be used to control gene expression with unprecedented precision--no other method offers such capabilities.

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.

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).View Large Image | View Hi-Res Image | Download PowerPoint Slide

A nanopore is the ultimate analytical tool with single molecule sensitivity. Going beyond analysis, synthetic biology also demand tools capable of reprogramming a cell's genetic code. Conventional electroporation lacks biomolecular specificity resulting in low cell viability and non-uniform transfection. We developed a single molecule specific and highly efficient strategy transfection single cells. Figure (a) illustrates the new nanopore-electroporation-tool (NEP): A confocal micrograph showing the silicon nitrate membrane with nanopore (b). The membrane separates the cis microfluidic channel from the trans. On the cis-side, 20 kbp double-stranded DNA intercalated with YOYO dye is conveyed via the microfluidic to the membrane. Inside the trans channel an E. coli cell is placed using optical tweezers in close proximity to the pore_so close that the field emanating from it affects cell membrane potential causing it to electroporate; consequently the bacteria fluoresces (a) and is subsequently encapsulated in hydrogel (c). With precise engineering of the geometry molecules such as ions, proteins or RNA can infuse into the gap between membrane and cell, or directly into the cell via electroporation. A pore so close to the cell offers the opportunity to both transfect the cell and measures its secretome simultaneously.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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.View Large Image | View Hi-Res Image | Download PowerPoint Slide

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.

Two sample cells designed specifically for sum-frequency-generation (SFG) measurements at the solid/liquid interface were developed: one thin-layer analysis cell allowing measurement of films on reflective metallic surfaces through a micrometer layer of solution and one spectroelectrochemical cell allowing investigation of processes at the indium tin oxide/solution interface. Both sample cells are described in detail and data illustrating the capabilities of each are shown. To further improve measurements at solid/liquid interfaces, the broadband SFG system was modified to include a reference beam which can be measured simultaneously with the sample signal, permitting background correction of SFG spectra in real time. Sensitivity tests of this system yielded a signal-to-noise ratio of 100 at a surface coverage of 0.2 molecules/nm(2). Details on data analysis routines, pulse shaping methods of the visible beam, as well as the design of a purging chamber and sample stage setup are presented. These descriptions will be useful to those planning to set up a SFG spectrometer or seeking to optimize their own SFG systems for measurements of solid/liquid interfaces.

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.

Understanding the organization and orientation of surface-immobilized single stranded DNA (ssDNA) in aqueous environments is essential for optimizing and further developing the technology based on oligonucleotide binding. Here the authors demonstrate how sum-frequency-generation (SFG) spectroscopy can be used to compare the structure and orientation of model monolayers of ssDNA on gold in air, D(2)O, and phosphate buffered saline (PBS) solution. Films of adenine and thymine homo-oligonucleotides showed significant conformational changes in air versus aqueous environments in the CH stretching region. The thymine films showed changes between D(2)O and PBS solution, whereas the SFG spectra of adenine films under these conditions were largely similar, suggesting that the thymine films undergo greater conformational changes than the adenine films. Examination of thymine films in the amide I vibrational region revealed that molecules in films of nonthiolated DNA were lying down on the gold surface whereas molecules in films of thiol-linked DNA were arranged in a brushlike structure. Comparison of SFG spectra in the amide I region for thiol-linked DNA films in air and D(2)O also revealed substantial conformational changes.