NANOBIOTECHNOLOGY
Nanobiotechnology is a rapidly advancing area of scientific and technological opportunity that applies the tools and processes of nano/microfabrication to build devices for studying biosystems. Researchers learn from biology to create new micro-nanoscale devices to better understand life processes at the nanoscale.
The Vision
Nanobiotechnology is beginning to generate substantial new insights into how biological systems function, and likewise, nanobiotechnology will lead to the design of entirely new classes of micro- and nanofabricated devices and systems.
The use of microfabrication as a method of miniaturizing biological and biomedical devices is just beginning to reach the biotechnology industrial community. Compared to the electronics industry, the fabrication technology now employed in biotechnology industrial development is relatively unsophisticated. This is due, in part, to the challenge of the vastly more diverse array of materials and chemical systems important to biological applications, compared to silicon-based technology in the integrated circuit industry.
Thus new fabrication processes must be developed for use with biologically relevant material systems. At the same time, the ability to effectively address dimensions at the molecular scale will open a new world of understanding and methods for scientific exploration and device construction.
Shrinking the Samples
Nanobiotechnology involves making small things and also working with them. Biological samples of interest contain ever smaller numbers of cells. This introduces new challenges to the preparation and analysis of these minute samples. Although some techniques readily meet these challenges, others could not—until recently—accommodate very small volume samples.
"You have been able to make many measurements from small volumes with mass spectrometry," says Charles Robertson, chief technology officer at NanoDrop Technologies, "but for these small samples, there was no way to make optical measurements—the primary means of obtaining critical quantitation data."
To solve that dilemma, Robertson and his colleagues developed spectrophotometers that work on microliter samples. This company offers shoebox-size spectrometers that work in the ultraviolet-visible range and fluorospectrometers, too. According to Lynne Kielhorn, NanoDrop's business development director, "We often talk about our instruments as enabling the growth of microgenomics." For instance, by using laser-capture microdissection, scientists can grab a small number of cells of interest, but then they need tools for preparing and analyzing these minute samples. "The big hole," she says, "was the ability to know the quality and purity of the sample as it was being processed. Our instrument can do the quality control from the beginning of taking a sample through preparation for microarrays."
The NanoDrop spectrophotometers simplify such measurements. Joel Hansen, NanoDrop's technology director, explains: "Rather than a cuvette or capillary, surface tension holds the sample in our device. Imagine holding a drop of water the size of a pinhead between your thumb and finger. It stays attached to both surfaces."
The same approach holds a microliter sample in the NanoDrop spectrophotometers, but the surfaces are the tips of optical fibers, leading to a light source and a detector. So a scientist pipettes a one microliter sample onto a surface of the spectrophotometer, and it makes the measurement and analyzes the sample. "You can fairly comfortably analyze three samples a minute," says Hansen. In a similar manner, NanoDrop's fluorospectrometer provides emission spectra of one-microliter samples. "It uses three LEDs: ultraviolet, blue, and white," says Kielhorn. Uniquely clean optics and virtual filtering enable the use of the broad spectrum white source, "so there is no need for filter changes or the expense of a monochromator," says Kielhorn. "It can also run samples with multiple fluorophores." It also does all this at high sensitivity. "It can detect just two picograms of double-stranded DNA," Robertson says. "Keep in mind that a human cell only contains three picograms."
Hansen notes that scientists already use the spectrophotometers in many applications, including normalizing templates for quantitative PCR, developing probes for microarrays, histocompatability for organ transplants, forensic analysis, and much more.
Speeding Up Sequencing
Nanotechnology also picks up the speed of genome sequencing. For example, 454 Life Sciences uses a nanotechnological approach to sequencing in which one instrument produces more than 20 million nucleotide bases in a four hour run—more than 100 times the capacity of instruments using the current macro-scale technology.
Likewise, Nanosphere makes a system that automates the detection of nucleic acids and proteins by using gold nanoparticles. "With nanoparticle probes," says Bill Moffitt, chief executive officer of Nanosphere, "you do not need to manipulate the sample to increase the abundance of the target. Our nanoprobes are also stronger reporters than fluorophores or chemiluminescent probes." In addition, Bill Cork, Nanosphere's chief technology officer, points out that their probes—with about 200 oligos attached to each of them—are extremely sensitive. For example, these probes can detect a single nucleotide polymorphism, or SNP, in the human genome. This system also provides very sensitive detection of proteins. "Today's protein assays work with concentrations of about 50 picograms per milliliter," says Cork, "and we are around 50 femtograms per milliliter."
Moreover, the Nanosphere probes get packaged in a disposable microarray. "That way," says Moffitt, "scientists do not need to mix master buffers or probe solutions. It is so simple to use that it could go in any doctor's office." As Cork explains one example: "You put DNA in a hybridization unit, read a barcode on the sample, put the unit in the processor, and it runs the entire assay—detecting all of the spots automatically—in just 60 to 90 minutes." This device can also multiplex. For example, Nanosphere's cystic fibrosis cartridge includes 26 SNPs.
Enhanced Imaging
Nanobiotechnology also requires ways to see molecular—and even atomic—features. An electron microscope, for example, can provide 0.1 nanometer resolution. Transmission electron microscopy (TEM) is based on the passage of electrons through a stained ultrathin section of material. Scanning electron microscopy (SEM) permits the three-dimensional view of a specimen's surface. Companies like Jeol, Hitachi, and Carl Zeiss produce electron microscopes for life science research.
One exciting advance takes SEM to wet samples. "By wet samples," says Dirk Stenkamp, managing director of Carl Zeiss' nanotechnology systems division, "we mean ones that are more or less embedded in their natural environment." This requires special vacuum conditions. "You run the sample and the chamber at a pressure that is above the water triple point, meaning you can still have bubbles or water condensed around your sample, which enables studying materials in their original aqueous environment," explains Stenkamp. Surprisingly, this system is also easy to use. "You just open the airlock of the system," says Stenkamp, "put in the sample, make two or three mouse clicks to close the airlock, set the pressure you want, and start looking at the sample."
Scientists can apply electron microscopy to many areas of nanobiotechnology. For example, TEM reveals the atomic structure of carbon nanotubes, and it can chemically analyze materials. SEM can image virtually any biological material and even features of semiconductors, such as transistor gates. In addition, Zeiss recently acquired ALIS Corp., a company that developed a completely new form of microscopy, which creates images with helium atoms instead of electrons or photons. "This provides higher resolution than SEM," says Stenkamp, "and it provides better contrast in biological materials."
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Focusing with Force
In some cases, scientists can zoom in even closer with atomic force microscopy (AFM). In this technology, an atomically sharp tip is raster-scanned over a surface to produce a three- dimensional representation of the surface. By vertically moving the tip into and out of contact with the surface, AFM can also provide quantitative measurements of tip-surface interaction forces. Applications of AFM in cell biology—from companies like Jeol and Veeco Metrology Group—include analysis of nucleic acids, cellular membranes, and proteins.
"AFM lets you look at biological interactions in real time, in situ, and under near physiological conditions," says Andrea Slade, life science applications scientist at Veeco. In fact, Veeco makes AFMs for many biological applications, such as the MultiMode scanning probe microscope for especially high resolution imaging and the new Bioscope II. Nelle Slack, life science applications scientist at Veeco, says,
"The Bioscope II is completely compatible with the majority of inverted optical microscopy techniques." In fact, the Bioscope II can add AFM capabilities to any optical scope. "You can run AFM while running the inverted microscope," says Slack. "You can look at two length scales simultaneously." That provides optical resolutions of a couple hundred nanometers and AFM resolution of a few nanometers, or even better in some cases.
Life scientists already use AFM for many purposes. Slade says, "AFM can reveal reorganization of membrane receptors after binding events or show how cells respond to perturbations in their local environment."
She adds that one research group even combined AFM and optical microscopy for nanosurgery—using the AFM tip to inject particles into specific cells and then optically observing the particles with fluorescence.
Shrinking Probes
In many experiments, watching interactions depends on attaching probes to specific molecules. Companies like Evident Technologies and Invitrogen (which recently acquired Quantum Dot Corporation) are creating smaller and smaller probes, such as quantum dots, which are nanosize crystals that can emit many different colors.
Evident Technologies, for example, developed EviFluors, which are antibodies conjugated to quantum dots. These nanoparticles can be used with fluorescence microscopy to study the subcellular trafficking of signals and the regulation of cellular functions. Jeff Goronkin, vice president of life sciences at Evident Technologies, says, "Using one ultraviolet excitation source, quantum dots can multiplex, or track a number of different proteins in one sample." Quantum dots also resist bleaching so they can track targets for longer periods of time.
These probes also offer a wide range of applications. "Potentially, any place that you can use fluorescence, you can use quantum dots," says Goronkin. The quantum dots will also move nanobiotechnology into new areas. For example, Evident recently received a grant from the U.S. National Institutes of Health to develop a quantitative assay for multiple breast cancer markers. He adds, "We're just scratching the surface of what quantum dots can do."
Nanobiotechnology, too, will surely delve much deeper into the life and clinical sciences. As assays and tools grow smaller, scientists will explore finer details of biological processes and build smaller tools to explore areas where science could not go in the past. This fantastic voyage is only getting started.
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BEYOND BIOLOGY
As if the blueprint for life wasn't busy enough, nanotech researchers are putting DNA to work in tiny mechanical devices and as templates for electronic circuits. Recent DNA constructions include microscopic patterns, tiny gears and a molecular assembly line. Although still mostly at the demonstration level, DNA nanotech is a rapidly growing field.
The first person to see DNA's potential beyond biology was Naiman Seeman, a chemist at New York University. More than twenty years ago, he began imagining how the genetic information in DNA might be engineered to perform useful tasks.
"DNA structures are programmable by sequence, and so are their intermolecular interactions," Seeman says. "That makes them unique."
Whereas nature alone dictates how most molecules interact, DNA comes with a built-in code that researchers can re-formulate to control which DNA molecules bond with each other. The goal of this DNA tinkering is microscopic factories that can produce made-to-order molecules, as well as electronic components 10 times smaller than current limits.
"Nanofabrication is where we are going," Seeman says. "It will happen soon."
Smart Glue
A single strand of DNA is essentially a long sequence made up of the chemical bases adenine (A), thymine (T), cytosine (C) and guanine (G). Every living thing carries a unique genetic code in its cells written out in these "letters."
Two strands of DNA can fuse together and form the famous double helix, discovered by Crick and Watson in 1953. But this twisted ladder arrangement can only happen if all the bases on the two strands match up, so that A's bond with T's and C's bond with G's. Scientists use this selective adhesive to build and control DNA machines.
"The bonds are like smart glue that know which pieces go together," explains Thomas LaBean of Duke University.
LaBean and others typically begin with a design for a structure that has several DNA pieces. A computer program writes out the code for the different strands, which are then synthesized using standard biological methods. Mixed together in a water-based solution, the pieces with matching codes will link up to form several copies of the desired structure.
It's like an airplane model kit, except all you have to do is shake the box and all the little parts automatically find each other and glue together.
Puzzle Pieces
DNA in nature is often just one long continuous chain, but researchers would prefer to have other shapes at their disposal.
More than three decades ago, biologists discovered that cells create cross-shaped DNA molecules during replication and repair. The side-arms, or branches, grow out of a genetic code whose letters read the same forwards and backwards, like the palindromes "racecar" and "rotator."
Tiny Rotator |
Recent research has shown that the length of palindromic DNA molecules can be controlled by rotation.
This is the first time that these symmetric molecules have been manipulated," says physicist Francois Heslot. "They react like a small molecular gear.
Heslot and his team at the Ecole Normale Superieure in Paris, France, attached a magnetic bead to one end of a long double strand of palindromic DNA. By applying a rotating magnetic field, the researchers untwisted the double helix, causing it to buckle in the middle. The resulting cross-shaped molecule began to shorten as the scientists unraveled it more. Approximately 200 full turns corresponded to a decrease in length of one micron (one millionth of a meter).
By reversing the turning direction, the palindromic DNA grew back in length, implying that it could be used to convert rotation into translation in a mechanical nanodevice, the authors speculate. |
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Seeman and others have modified the sequence of palindromic DNA to make a stable 4-armed molecule. They have also coaxed DNA to branch with 3, 5 and 6 arms. These two-dimensional stick figures are only a few nanometers across, where a nanometer is one billionth of a meter. Researchers design them with "sticky ends"—single DNA strands that act as latches between molecules. Whole arrays of these connecting figures can be put together like pieces in a puzzle.
Earlier this year, LaBean and his collaborators built 4x4 lattices with 16 cross-shaped DNA pieces. By attaching a type of protein to specific "pixels" on these grids, the team spelled out "DNA."
The ability to attach particles to DNA pieces is a step towards fabricating nano-electronics. Scientists can hitch functional materials like metals, semiconductors and insulators to specific DNA molecules, which can then carry their cargo to pre-specified positions. Already this technique has been used to make a simple transistor, as well as metallic wires.
There is a problem, however, in making more complicated components. To keep negatively-charged DNA stable, researchers add positive ions to their solutions. But these ions can interfere with the functional materials needed to build electronics.
"It is difficult to keep all these things happy at the same time," LaBean says.
A solution might be to use a DNA-like molecule that is uncharged and yet has the same code as DNA. There are about 1000 "flavors" of DNA derivatives, Seeman says, so one of these might do the trick.
Trouble is these alternatives can be 10 times more expensive to make than regular DNA, according to LaBean. It could be worth it, however, as computer chip manufacturing techniques currently cannot go smaller than tens of nanometers.
Self-assembling arrays of DNA-like molecules could go beyond this limitation, by providing the scaffolds for nanometer-scale circuits. This would not only make our computers and other devices more compact, but faster as well.
Nano Robots
Besides controlling the shape of DNA assemblages, researchers can use specific DNA attachments to move other DNA molecules.
One of the first demonstrations of this came in 2000, when a group from Lucent Technologies in New Jersey fabricated a short V-shaped DNA molecule that acted like molecular tweezers.
Placing several copies of their molecule in solution, the researchers could snap the tweezers shut by mixing in another DNA molecule, called a "set strand," that bonds specifically to the two ends of the "V" and pulls it closed. To reopen the tongs, the science team added an "unset strand," which links to the set strand and pulls it off the tweezers.
Using a similarly orchestrated movement, Seeman and his colleagues in 2004 made a two-legged DNA molecule that could walk. The feet were anchored to a DNA-studded floor by set strands. The tiny biped took a step whenever the group introduced unset strands that freed one leg at a time.
Assembly Line
More recently, Seeman and colleagues have put DNA robots to work by incorporating them into a self-assembling array. The composite device grabs various molecular chains, or "polymers," from a solution and fuses them together. By controlling the position of the nano-bots, the researchers can specify the arrangement of the finished polymer.
Seeman hopes this tiny assembly line can be expanded into nano-factories that would synthesize whole suites of polymers in parallel. The major challenge now is going from 2D arrays to 3D structures. The extra dimension would allow the fabrication of more elaborate molecules, as well as denser electronic circuits.
In the future, doctors might inject variants of these automated DNA machines into the body, either as bio-sensors or as drug delivery systems that can target specific sites like tumors or blood clots, LaBean said. Although some of these applications may be several years down the road, progress in DNA nanotech
"has become a lot faster now that there are 20 or more groups doing it rather than just my own," Seeman said.
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