DOI: 10.1126/science.opms.p0600010

Life Science Technologies
Nanobiotechnology:
An Incredible Voyage for the Life Sciences

Shrinking the size of devices to the atomic scale promises advances in basic and applied biological research. Many disciplines—from cell biology and chemistry to engineering and physics—coalesce in advanced forms of assays, automation, and medicine. The nanometer scale of these products offers exquisite control of experiments and new tools for basic and applied research.

By Mike May and Gary Heebner

Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.

Defining nanobiotechnology seems easy enough: technological applications on the billionth of a meter scale related to biology. Most scientists in this field would probably agree with that definition, but there are twists, too. Christine Peterson, vice president and co-founder of the Foresight Nanotech Institute, calls it "the interactions of biological materials or systems with nanotechnological materials and devices." Nonetheless, she adds that the description of this field continues to evolve.

Instead of focusing on what is or is not part of nanobiotechnology, scientists wonder more what is going on in this broad area. First, this field brings researchers together from many areas: cellular and molecular biology, chemistry, engineering, physics, and more. In addition, nanobiotechnology aims at improving automated laboratory procedures, imaging, diagnostic assays, and more. In the near term, says Peterson, "The most exciting developments will probably be in cancer treatments. Some wonderful results are already coming from that area." She also expects nanobiotechnology to trigger advances in the early detection of a variety of diseases and improvements in biological implants.

Peterson adds that these are early days for nanobiotechnology, but she says that "the funds are already flowing." As a result, she expects even more benefits in the next decade. For example, she mentions faster DNA sequencing. "You hear predictions of rapid DNA readings in doctors' offices," she says. On an even more distant horizon, she believes that microscopic robotic devices will emerge from nanobiotechnology. "Scientists want to combine the chemical action of drugs with the three-dimensional control of surgery," Peterson says. "The ultimate goal will be to work at the molecular level with nanoscale devices."

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.

Microscopic Currents

As technology addresses small scales, fluids push through smaller channels—even nanoscopic channels, such as carbon nanotubes. In microfluidics or even nanofluidics, scientists treat the surfaces of these devices or pack them with chromatographic media to separate a mixture of molecules. A number of companies—including Gyros and Nanosys—make systems for manufacturing such devices.

Such miniaturization can improve traditional techniques, such as high pressure liquid chromatography, or HPLC. For example, Nanostream introduced micro parallel liquid chromatography, which allows simultaneous chromatographic analysis of many compounds. Eksigent Technologies also provides products for nano HPLC and pumping nanoscale volumes.

Likewise, Agilent developed its microfluidics-based HPLC-Chip/MS technology. This system combines nanoflow HPLC columns, associated connections, and a nanospray emitter on a reusable microfluidic chip. Georges Gauthier, HPLC-Chip technology product manager at Agilent, says, "This helps people who work with very small sample sizes or very dilute samples, and need high sensitivity." He adds that the simplicity of this system "moves microfluidics into day-to-day use."

A scientist puts a sample on the microarray, and the instrument performs the chromatography and mass spectrometry. This system can even use very dilute samples, such as proteins in the attomole (1 x 10^-18 moles) range. The chip incorporates an enrichment step, and the customer can pick the injection volume. So far, scientists have applied this system in a variety of applications, including searching for biomarkers and identifying proteins, or even modifications to proteins, such as phosphorylation and glycosylation.

Nanodetection

The nanorange also allows new approaches to analysis, such as nanoelectronic detection. David Macdonald, chief executive officer of Nanomix, says, "This harnesses the size and semiconductor characteristics of carbon nanotubes to detect things electronically rather than optically." For example, Macdonald's company uses a network of carbon nanotubes that are functionalized with specific chemistries. "Then, we can detect a variety of gases and biomolecules," says Macdonald, "all using the same device but with different chemistries and no labeling."

One key to this approach comes from the size of the carbon nanotubes, which are only a nanometer or two in diameter. "This makes them very sensitive to hybridization and binding on their surfaces," says Macdonald. "Even with a low concentration of analyte, we get a large electronic response." Perhaps better still, it can all be packaged in arrays. "We can reduce benchtop equipment to a handheld, disposable device—just the size of a cell phone," Macdonald explains.

Nanomix started with a hydrogen sensing product. Now, the company is working on other applications, including a disposable capnometry device, which measures exhaled carbon dioxide and can be used anywhere to assess a patient's breathing. To expand the applications of nanodetection, Nanomix also collaborates with other companies. "We have entered into several joint development agreements where we collaborate on product development and give exclusive distribution rights through a supply agreement," says Macdonald.

Adding to Arrays

In the last decade, many cellular and molecular biologists turned to microarrays, from companies like Affymetrix, Agilent, Ciphergen, and NimbleGen. Recently, Affymetrix joined forces with Caliper Life Sciences to develop hybrid microarray-microfluidic systems that can industrialize genomic research.

In addition, microarrays can be used in preparation for other technologies. Jordan Stockton, marketing manager of informatics and gene expression at Agilent Technologies, says, "Microarrays can be used as precursors to nanotechnology experiments or as follow-ups." He says that scientists can use microarrays to survey whole genomes or segments of a genome for global patterns. Then, they might turn to nanotechnology to focus on areas of interest.

At Agilent, ink jet printers spray spots onto the microarrays. Stocton says, "With our piezoelectric ink jet technology, we print nucleotides and incorporate them in growing oligos on a slide. It's microfabrication." Moreover, customers can upload sequences online to design the specific microarray that they desire.

Using this technology, Agilent produces a variety of products. For example, scientists can buy microarrays that contain four entire genomes on a single slide. "Gene expression data are inherently noisy," says Stockton, "so running replicates per sample is crucial. Now, you can do that conveniently and economically." Instead of putting four genomes on a microarray, scientists could also print eight subgenomes to simultaneously run even more replicates. In addition, Agilent's microarray-based Comparative Genomic Hybridization (aCGH) includes 240,000 spots, which "let scientists look at single copy number changes across the genome," says Stockton. Moreover, this company's ChIP-on-chip product looks at potential binding sites for transcription factors. "It can track where individual transcription factors bind to chromatin," says Stockton. "It is actually probing how chromatin changes and how that drives downstream transcription changes." He adds, "Once scientists see where a transcription factor binds, nanotechnology—like labeled beads—can be used to look even closer."

Microarrays with so many spots, followed with nanotechnological approaches, generate lots of data. Then, scientists need some way to compare all of the data. "You try to triangulate the results," says Stockton. "You want to glue all of the results together." Agilent scientists are working on software tools that, says Stockton, "are intelligent enough to make educated guesses about what types of measurements are related and then depict them graphically."

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."

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.

Mike May(mikemay@mindspring.com) )is a publishing consultant for science and technology based in the state of Minnesota, U.S.A. Gary Heebner (gheebner@cell- associates.com) is a marketing consultant with Cell Associates in St. Louis, Missouri, U.S.A.

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