HOW NANOTECHNOLOGY WORKS

INTRODUCTION
Because elements at the nanoscale behave differently than they do in their bulk form, there's a concern that some nanoparticles could be toxic. Some doctors worry that the nanoparticles are so small, that they could easily cross the blood-brain barrier, a membrane that protects the brain from harmful chemicals in the bloodstream. If we plan on using nanoparticles to coat everything from our clothing to our highways, we need to be sure that they won't poison us.
Closely related to the knowledge barrier is the technical barrier. In order for the incredible predictions regarding nanotechnology to come true, we have to find ways to mass produce nano-size products like transistors and nanowires. While we can use nanoparticles to build things like tennis rackets and make wrinkle-free fabrics, we can't make really complex microprocessor chips with nanowires yet

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Methods

Design, fabrication and cell-handling of microfluidic device for single cell electroporation The silicon-glass microfluidic device contains two channels that are connected by microholes, which act as trapping sites for living cells (Fig 1a–b). The two microfluidic channels (widths of 50 µm and 20 µm, respectively) and the nine trapping sites (width 4 µm) are etched in the top side of a silicon wafer by reactive ion etching (RIE–depth 15 µm). Fluid reservoirs and connections to the channels are powderblasted from the backside of the silicon, followed by thermal oxidation of the silicon (318 nm) to electrically isolate it from the metal electrodes. The channels are closed with a glass wafer (Pyrex), which allows the visualization of the trapping and the electroporation process. Platinum electrodes are sputtered onto the Pyrex prior to anodic bonding to the silicon wafer with the fluidic structure, after which the wafer stack is diced into individual chips.

Fig. 1 Silicon-glass microfluidic device for single cell electroporation and gene transfection studies (20 × 15 × 1 mm) (a) Artistic 3D impression of trapped cells; (b) Microfluidic chip layout, zoom-in on trapped single cells; (c) Analysis of the electric field strength distribution at the trapping sites shows that a voltage of 1 V yields an electric field strength of 0.57 kV cm–1.
The electrodes are positioned such that individual traps can be electrically addressed. The electric field is focused at the trapping sites (Fig 1c), while it is negligible in the upper channel, as well as in the neighboring trapping sites (as analyzed with finite element modeling). Thus, cells which are not trapped or trapped at the neighboring trapping sites are not affected by the electroporation protocol, as was verified experimentally.
The integrated microholes structure in the flow-through chip represents a way of handling single cells. The cell sample flows along the upper channel, while the lower channel is used to create an under-pressure, which was accomplished by suction (via a pump, 1–2 psi) on the bottom access hole. Once the cells have been trapped, the pump is switched off and cells are localized at the traps.
After trapping of cells, the DNA construct to be electroporated is loaded into the cell trap device. 40 µl of the DNA construct at a concentration of 100 ng ml–1 is added in the top and bottom reservoirs and transported into the microfluidic channels by hydrodynamic flow. DNA–cell contact is established within a pre-incubation period of 10 min before applying an electric field. Subsequently, DNA transport across the cell membrane is initiated by one electric pulse of 6 ms and electric field strength of 0.67 kV cm–1. A post-incubation time up to 10 min is given for the cell–DNA mixture before cell culture medium is added to the microfluidic channels. The chip is then immersed in a small Petri dish with this cell culture medium and placed in the incubator (37 °C and 5% CO2). Optical inspection for gene expression of the cells is done 24 h after electroporation.

NANOTECHNOLOGY

Mouse myoblastic cells (C2C12)
C2C12 (ATCC) cells were cultured in DMEM medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 IU ml–1 penicillin, 100 mg ml–1 streptomycin, 2 mM L-glutamine, 250 mg ml–1 fungizone and 1% sodium pyruvate. Cells were harvested by incubation with 0.025% (w/v) trypsin (Invitrogen).
Mesenchymal stem cells (MSCs)
Mesenchymal stem cell growth medium consisted of MEM (Invitrogen), 10% FBS (Cambrex), 1 ng ml–1 bFGF (Invitrogen), 100 µg ml–1 penicillin, 100 IU ml–1 streptomycin (Invitrogen), and 0.4 mmol ml–1 ascorbic acid (Sigma). Cells were harvested by incubation with 0.25% (w/v) trypsin (Invitrogen). Cells were used up to passage number 6 (approximately 40 passage doublings)
For electroporation
Cells were harvested using 0.25% trypsin. Trypsin was inactivated by addition of complete growth medium, followed by centrifuging. Cells (C2C12 or MSC) were resuspended in electroporation buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.68 mM KCl, and 1.7 mM MgCl2, 25 mM glucose, pH 7.4) at a concentration of 0.5×106 cells ml–1.
Generation of an EGFP-ERK1 fusion construct
ERK1 DNA was amplified using PCR with Pfu Polymerase (Promega). The PCR primers were directed against the start and stopcodon and included specific restriction sites for easy isolation. The PCR products were cloned into PCR2.1 using a TOPO TA cloning kit (Invitrogen). ERK1 was isolated from PCR2.1 by digestion with XhoI and BamHI and ligated into the XhoI and BamHI sites of pEGFP-N1 (Clontech).
Instrumentation
The microfluidic device was mounted onto an X-Y-Z translation stage on an inverted wide fluorescence microscope (Leica DM IRM, Leica Microsystems, Wetzlar, GmbH, Germany). The microscope system is equipped with a mercury lamp, 20×, 40×, 50×, 63× objectives, and a fluorescence filter set (BP 480/40, LP 515). In addition, a computer-controlled CCD camera (Leica DFC300 FX) was mounted on the microscope for image recording. For the electroporation signal, a custom-made Labview (National Instruments) application controls a function generator card (NI5041, National Instruments) and a data acquisition card (NI PCI-6221, National Instruments), which were connected to the electrodes of the device via an especially home-designed holder.
Cell imaging and correction for photobleaching
Track images were taken every 5 min with 488 nm excitation and a LP 515 emission filter. Fluorescence intensity changes in either the cytoplasm or nucleus of the cells was determined using ImageJ software.13 For correction of photobleaching, it is assumed that over the time of the experiment, the total fluorescent intensity in the cell is constant. In order to determine the corrected intensity, the mean intensity is calculated inside a mask of the whole cell (I0) and inside a region of interest, defined manually, both in the cytoplasm and in the nucleus of the cell upon their position over time. Subsequently, each pixel in each frame was multiplied by the factor (I0/It), where I0 is the original intensity (t = 0) and It is the current frame intensity. For time zero (t = 0 min) the correction factor is 1, but as the image bleaches, the correction factor gets larger. This correction factor was applied to the track images for the nuclear translocation of EGFP-ERK1, as well as to the graphs of the fluorescence intensity versus time.
Electric field distribution
The electric field distribution in the cell trapping device has been modeled using finite element modeling software (Femlab 3.0, Comsol, Sweden) for a 2D situation. The DC conductive medium model was used for a static state situation, which solves Gauss' differential equation. The boundary conditions are considered as being electrically insulated, with the two electrodes being 1 V and grounded. The subdomain has a conductivity of 0.1 S m–1.
Results

Electroporation of C2C12 cells
In order to introduce DNA into cells, we determined the electrical parameters for electroporation of mammalian cells. Since human adult stem cells are relatively hard to isolate and cannot be kept in culture indefinitely, we used a C2C12 mouse myoblastic cell line that is similar in size and has some of the properties of the MSC.
The voltage (E-field) and pulse length needed for cell membrane permeabilization of individual C2C12 cells that are trapped in the cell trap device were determined by using propidium iodide (PI) as membrane integrity indicator. In a series of experiments, pulses with different amplitudes and/or pulse lengths were applied to individual cells and the fluorescence intensity of the cell was measured one minute after each single pulse. At the start of each experiment, trapped cells were not red fluorescent, indicating that the cell membrane was intact. First the minimum voltage for successful PI uptake was determined for a pulse length of 6 ms. Electrical pulses of 6 ms with increasing amplitude from 1 to 3 V (steps of 0.5 V) and an interval pulse of 1 min were applied to the cells (Fig. 2a–b and supplementary Fig. S1). Subsequently, for the lowest voltage for electroporation of C2C12 cells (2 V), the effect of the pulse length on PI uptake was studied (supplementary Fig. S2). In order to test DNA uptake under the defined parameters, a DNA construct encoding green fluorescent protein (EGFP-N1) was electroporated into C2C12 cells. Twenty-four hours after electroporation, EGFP expression was visible in all cells as green fluorescent staining (Fig. 2d and Table 1). The cells also showed healthy morphology observed under light microscopy images. This healthy morphology was maintained at least 120 h post-transfection (after which the cells had divided one time). Thus, a potential of 2 V for at least 6 ms results in single cell electroporation of all C2C12 cells. This electroporation procedure resulted for the first time in GFP transfection and expression into single C2C12 cells and was used to transfect plasmids into MSCs.

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Compatible Materials

Silanes
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Alkanethiol Monolayers
PDMS
Hydrogels
Nitrocellulose
Polystyrene

Technical Characteristics
Spot size: ultra-micro and sub-micron (femtoliter to attoliter)
XY Resolution: 20 nm
XY Travel: 25 mm
Z Clearance: 45 mm
Printing cycle: 100ms
Spot or Line Size: 1-60 um
Spot Volume Range: attoliter to femtoliter
Humidity Range: 25-80% RH
Optical Magnification: 150x – 1000x
Average Print Cycle: 100 msec

Benefits
Larger patterns: 62,500 times more XY patterning area than Scanning Probe Lithography systems
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APPLICATIONS

CURRENT APPLICATIONS:
Nanocomposites – materials created through the introduction of nanoparticulates into a macroscopic sample material for the purposes of introducing thermal and electrical conductivity and/or to increase the strength of the material.
Nanocrystals – nanoparticles with a crystalline structure ideal for harvesting light energy in photovoltaic devices.
Nanoparticles – microscopic particles used in the creation of common items as stain-proof fabrics, sunscreens, paints, and synthetic bone.
Nanotubes – nanometer scale wire-like material typically composed of Carbon.
Nanorobots – nano-scale machines programmed to perform specific tasks. Nanorobots may one day peform the functions of antibodies and cellular repair
fabrication technique:A new fabrication technique, known as soft interference lithography (SIL), makes it possible to inexpensively produce large sheets of gold films with virtually infinite arrays of perforations and microscale "patches" of nanoscale holes. A combination of interference lithography and soft lithography, SIL offers many significant advantages over existing techniques. It can be used to scale-up the nanomanufacturing process to produce plasmonic metamaterials and devices in large quantities. Devices such as films of nanoholes can also serve as templates to make their inverse structures, such as nanoparticles. (Legend: Si=silicon; Cr=chromium; PEEL=a fabrication procedure Odom and her colleagues developed to make free standing films.)

NANOTUBES

Nanotechnology is an emerging and promising field of research, loosely defined as the study of functional structures with dimensions in the 1-1000 nanometer range. Certainly, many organic chemists have designed and fabricated such structures for decades via chemical synthesis. During the last decade, however, developments in the areas of surface microscopy, silicon fabrication, biochemistry, physical chemistry, and computational engineering have converged to provide remarkable capabilities for understanding, fabricating and manipulating structures at the atomic level.
Research in nanoscience is exploding, both because of the intellectual allure of constructing matter and molecules one atom at a time, and because the new technical capabilities permit creation of materials and devices with significant societal impact. The rapid evolution of this new science and the opportunities for its application promise that nanotechnology will become one of the dominant technologies of the 21st century. Nanotechnology represents a central direction for the future of chemistry that is increasingly interdisciplinary and ecumenical in application.

As practiced at Rice University, there are three distinct nanotechnologies:
"Wet" nanotechnology, which is the study of biological systems that exist primarily in a water environment. The functional nanometer-scale structures of interest here are genetic material, membranes, enzymes and other cellular components. The success of this nanotechnology is amply demonstrated by the existence of living organisms whose form, function, and evolution are governed by the interactions of nanometer-scale structures.
"Dry" nanotechnology, which derives from surface science and physical chemistry, focuses on fabrication of structures in carbon (for example, fullerenes and nanotubes), silicon, and other inorganic materials. Unlike the "wet" technology, "dry" techniques admit use of metals and semiconductors. The active conduction electrons of these materials make them too reactive to operate in a "wet" environment, but these same electrons provide the physical properties that make "dry" nanostructures promising as electronic, magnetic, and optical devices. Another objective is to develop "dry" structures that possess some of the same attributes of the self-assembly that the wet ones exhibit.
Computational nanotechnology, which permits the modeling and simulation of complex nanometer-scale structures. The predictive and analytical power of computation is critical to success in nanotechnology: nature required several hundred million years to evolve a functional "wet" nanotechnology; the insight provided by computation should allow us to reduce the development time of a working "dry" nanotechnology to a few decades, and it will have a major impact on the "wet" side as well.
These three nanotechnologies are highly interdependent. The major advances in each have often come from application of techniques or adaptation of information from one or both of the others. This critical symbiosis can flourish at Rice, where we already have a world-class status in computation, in the biosciences, and in the physical sciences and engineering disciplines that underlie "dry."

THE TRIPLE BOTTOM LINE BLOG

Technophobia and Religion: Can Businesses Overcome the "God Gap"?
Maybe you noticed this curious item in the front pages of the March 3rd Business Week (no link to this story):
In a recent poll, only 30% of 1,015 Americans said they morally approved of nanotechnology--the engineering of matter at the molecular level to create everything from slice-resistant golf balls to cancer drugs. That's much lower than the results of similar surveys in key nanotech markets like Britain (54%), Germany (63%), and France (72%), notes Dietram Scheufele, a University of Wisconsin life sciences professor who ran the U.S. poll. He believes the approval gap is due to religion's "important role" in America. On both sides of the ocean, he says, many who identified themselves as religious objected to the idea of altering molecules.I found the story weird because I've heard so little in the mainstream media about nanotechnology--and certainly nothing that would stir fears of Frankensteinish nanobots running amok. (By contrast, genetically engineered foods have gotten a lot more negative publicity--to say nothing of the hotly controversial stem cell research.)When I did a little further reading on the poll, I became even more puzzled, especially when I read a comment from Professor Scheufele to the effect that the popular opposition to nanotechnology is not due to ignorance about the technology but rather to religiously-based objections: "The issue isn't about informing these people. They are informed."What does this mean? Are we to believe that there are large numbers of devout Christians who are deeply knowledgeable about nanotechnology and have developed thoughtful theological arguments against it? The idea struck me as rather implausible--so much so that I emailed Professor Scheufele for further information.The articles the professor linked me to did a lot to clear up the mystery. Turns out that one of the things Scheufele and his colleagues studied was whether knowledge about nanotechnology is correlated with positive attitudes toward it; and another was how religious belief affects this relationship. As explained in a forthcoming article in Public Understanding of Science, co-authored by Scheufele, Dominique Brossard, and Eunkyung Kim,
. . . highly religious respondents . . . showed the lowest levels of support for funding for nanotechnology; and being more knowledgeable about nanotechnology did little to influence their support for funding. For less religious individuals, however, our data showed a strong link between knowledge about nanotechnology and greater support for nanotechnology funding.In other words, to know about nanotechnology is to love it--unless you are religious, in which case you hate it whether you know anything about it or not. (I'm oversimplifying, but you get the point.)I find this story slightly depressing. It suggests that the U.S. contains, at the moment, a large, essentially irreducible minority of people who reflexively oppose new technologies, fear them, and express these fears in moral terms. For these people, scientific innovation is equated with "playing God" or "tampering with nature," and they assume that there is something sinister about technology unless the opposite can somehow be convincingly demonstrated.The apparent link between technophobia and religious sentiment suggests specific issues that business people need to address.If you're in charge of public relations at Monsanto, Dow, Schering-Plough, Amgen, or any other company that is in the business of marketing technological breakthroughs, you ought to consider creating a department for religious outreach. Talk to theologians, including some conservative ones, and work on developing an appropriate, non-scientific, spiritual language for explaining what you do--perhaps describing technology in terms like "human stewardship of creation" and its benefits as "blessings produced by God's gift of reason."And devote resources to making connections with religious leaders, especially in the United States. When future public battles are fought over issues like technology regulation and funding, you'll probably want to have some well-informed, open-minded, and widely-trusted pastors on your side.