Nanobiotech Makes the Diagnosis

Gazing at an electrical meter, Yi Cui, a graduate student in the Harvard University lab of chemist Charles Lieber, waits for evidence of a remarkable feat in simple, ultrasensitive diagnostics. His target is prostate cancer. His new tool is a microchip bearing 10 silicon wires, each just 10 nanometers (billionths of a meter) wide. These nanowires have been slathered with biological molecules with an affinity for PSA, a protein all too familiar to men of a certain age as the telltale sign of prostate cancer. If the experiment works according to plan, when the PSA molecules bind to the nanowires, there will be a detectable electrical signal.

Cui washes a solution containing prostate cancer proteins over the chip. Immediately, the meter registers subtle changes, indicating not only that the device has detected the protein, but that it detected perhaps as few as three or four molecules, instantly and with minimal sample preparation-a previously unheard-of feat. The implications for diagnostics are enormous. A successful prostate cancer test must distinguish between normal and elevated protein levels. Ultrasensitive sensors like Lieber’s could discern the slightest increase; what’s more, they could do so in cheap, disposable tests that patients could use at home between visits to the doctor. “If I were at risk for a particular cancer, I wouldn’t want to take a chance and wait for some cancer cells to grow wildly out of control over a year because the previous test missed it,” says Lieber.

Though this nanowire device is just an experimental prototype, it is at the forefront of a growing effort at labs around the world to marry nanoelectronics and biology into a new field called nanobiotechnology. This hybrid discipline is producing a variety of tools-from arrays of tiny sensors that can detect specific biological molecules to microscopic systems carved out of silicon that can read individual strands of DNA-capable of providing a new window on biological molecules.

The implications for medicine and biotechnology are myriad. Besides sniffing out the barest whiffs of disease-or perhaps detecting a single spore of anthrax-these devices could provide far faster and easier diagnosis of complex diseases. For example, they could provide early warnings about heart attacks, whose calling cards are subtle changes in the mix of dozens of proteins. Alternatively, a single microchip could provide a comprehensive diagnosis from a drop of blood. And for drug researchers, nanobiotech gadgets could mean new tools for discovering and evaluating potential drugs more rapidly, by screening millions of different drug candidates at once. Some of these more ambitious goals will likely take years to achieve, but nanobiotech could lead to real devices that will begin replacing cumbersome lab-based procedures with cheap, accurate microchips in as little as two years.

These first products-chips rigged to detect a specific disease or cluster of genetic disorders-are already being developed at nearly a dozen nanobiotech startups (see ” Sensing Success “) . Larry Bock, CEO of Palo Alto, CA-based startup Nanosys [ TR board member Robert Metcalfe is a Nanosys cofounder and director. Ed.], which has licensed Lieber’s technology, predicts his company will market a commercial sensor within three years, first for use as a research aid to rapidly screen potential drugs, and later as a cheap, disposable at-home test for prostate cancer and perhaps other cancers. “People talk about all the wonders of nanotechnology but then say it’s not going to happen for another 20 years,” says Chad Mirkin, a chemist and director of the Institute for Nanotechnology at Northwestern University. “But that’s absolutely incorrect for things like diagnostics. You’re going to see products on the market in the next two years.”

Power in Numbers

Biology and electronics have long existed in separate universes. But because biological molecules, like DNA and proteins, are roughly a few nanometers in size, and because physicists and chemists are now learning how to make electronic devices on exactly that size scale, these universes are colliding. The result is a new class of devices that combine the ability of biological molecules to selectively bind with other molecules with the ability of nanoelectronics to instantly detect the slight electrical changes caused by such binding. “What’s really interesting about this technology is that it allows one to take the inorganic components that normally would be nestled inside an electrical chip and combine them with biological molecules,” says Paul Alivisatos, scientific cofounder of Nanosys and a chemist at the University of California, Berkeley.

Indeed, nanoelectronic devices like the one built in Lieber’s lab (see ” Sensitive Wire “) could do away with the elaborate apparatus now needed for ultrasensitive detection. “If you wanted to do single-molecule detection in a lab today, you would need a laser the length of a desk and a lot of sophisticated optics, chemical labels to amplify the signal enough to be able to see it,” Bock says.

Shrinking down such ultrasensitive devices enough that they could be put on chips could have numerous applications in diagnostics. Stanford University chemist Hongjie Dai, for example, has built a device that can detect glucose with a single carbon nanotube, a large carbon molecule with excellent electrical properties (see ” The Nanotube Computer ,” TR March 2002) . The glucose molecules react with molecules on the surface of the nanotube, creating electrical signals that correspond to glucose concentrations, he says. Though only a proof of concept today, such a device could be developed into an implantable glucose sensor for diabetics. In December, Dai launched Molecular Nanosystems in Palo Alto, CA, to commercialize nanotube-based devices including biosensors.

For many applications, though, what’s really needed is not a lone nano detector but a dense array of them. That way, you can rapidly look for thousands, even millions, of different biological molecules in a single drop of blood or other body fluid, allowing the diagnosis of diseases that have complex molecular signatures. One such disease is rheumatoid arthritis-an autoimmune disease with many variants, each marked by subtle differences in groups of proteins. Ideally, each variant would be fought with a slightly different treatment; in practice, sufferers today are generally treated in the same way. But, says Dai, a nano array could serve as a highly precise and discriminating diagnostic device, providing a road map for custom treatment.

These arrays of nano detectors promise advantages over existing technologies, like DNA chips, and ones under development, like protein chips. All such chips require fluorescent labeling of molecules and optical microscopes to detect the glow given off when binding occurs (see ” DNA Chips Target Cancer ,” TR July/ August 2001) . What’s more, roughly a thousand molecules must bind to each sensing element to create the glow. With nanoelectronics, no bulky, expensive equipment is needed, and instant detection of just a few molecules is possible.

Sensitive Wire

To detect a disease-related protein in a blood sample, a silicon wire just 10 nanometers wide is coated with biomolecules that bind only to that protein (below). When the disease protein binds to a molecule on the wire (inset), the wire’s conductance changes, providing an instant electric signal.

Sticky DNA

But sensors with nanoscale features can only succeed if they are “sticky” enough to grab onto molecules of interest. Northwestern’s Mirkin sees value in gold: specifically, nanoscale gold particles, to which he affixes multiple fragments of DNA that can latch onto DNA targets. Each gold particle becomes “like Velcro,” he says. In the next 18 months, Mirkin says, he and his colleagues will build a simple, doctor’s-office diagnostic device capable of instantly diagnosing diseases or predispositions to disease, depending on what DNA fragments are used on the device. “Chips will be built for panels of diseases,” says Mirkin, including sexually transmitted diseases, cystic fibrosis and genetic predispositions to colon cancer and blood hypercoagulation (blood that clots excessively).

Mirkin’s prototype chip, under development by Northbrook, IL-based Nanosphere, a company he cofounded, uses DNA deposited between electrodes on a microchip to recognize targets of interest. A sample is mixed with those “Velcro” gold particles and washed over the chip. If the sample contains the targeted DNA-say, genetic material from the syphilis bacterium-the DNA will bind to those sticky gold particles and then to the DNA fragments between the electrodes. The gold particles close the circuit and produce a detectable signal. The more electrode sensing elements per chip, the more diseases-or genetic predispositions-can be detected.

Mirkin’s group is adapting a process known as dip-pen nanolithography to gain the ability to literally “print” DNA molecules between electrodes just 200 nanometers apart. Mirkin hopes to pack hundreds, even thousands, of electrode sensing elements on one chip.

Printing Molecules In dip-pen nanolithography, molecules are printed directly on a chip surface.

Arrays of cantilevers (above) deposit millions, even billions, of different molecules on a surface; in cases where the printed molecules bind to specific genes or proteins, the chip can be used to diagnose diseases or discover drugs. Each cantilever, or “pen,” has a silicon tip (left) just a few atoms wide at its end. As the tip moves laterally, molecules attached to its sides are drawn down to the surface by a water meniscus that forms under the tip. The vertical motion of each cantilever is controlled thermally, allowing individual pens to start and stop printing.

Mirkin’s technology can find specifically targeted DNA in a sample. But if you could actually grab a single piece of DNA and directly “read” its genes, you could, in theory, identify any gene, or even complex gene patterns. Using tools adapted from semiconductor manufacture, physicist Harold Craighead of Cornell’s Center for Nanobiotechnology and his former postdoc Stephen Turner built a silicon chip containing tiny channels, each 50 nanometers in width and depth (see ” DNA Pipeline ,” below) . The channel is so small that a single strand of DNA can barely squeeze through-and that’s just the point. An electric field causes the normally coiled ball of DNA to bump into the channel, uncoil and thread its way down.

Once grabbed, the DNA needs to be “read”-to see, for example, if it contains a specific sequence. To make a sequence legible, researchers add fluorescent-labeled DNA probes to the sample beforehand; the probes bind to the target sequences. As each molecule of DNA wiggles its way down the channel, an optical detector identifies the fluorescent labels passing by. “We’re treating the DNA like it’s a recording medium,” says Turner, who is now president of Nanofluidics, a startup trying to commercialize the Cornell technology. “And just like a tape player, we’re playing the DNA.” While the Cornell researchers currently use an external optical microscope to read the “tape,” they hope to build an optical reader directly onto the chip using optical fibers. Turner expects to have a working device within the next few years.

Because the tools for making these tiny channels rely on the same standard equipment used to fabricate silicon chips for microelectronics, Turner envisions making nanofluidic chips with thousands and even millions of channels and optical fibers. With such devices, Turner says, doctors could one day take a drop of blood from a patient, drop it on the microchip and rapidly scan the DNA in the sample for genetic markers of disease. The device could also help doctors choose just the right drugs for the patient.

DNA Pipeline

To identify a particular sequence on a strand of DNA, researchers first mix the DNA with fluorescent probes that attach to that sequence. Then, on a microchip (above), an electrical field draws DNA through a channel 50 nanometers wide. An embedded optical reader detects any attached probes, identifying the sequence.

DNA Control

In the marriage of nanoelectronics and biology, the most extreme vision involves affixing electronic gadgets directly to molecules. To show how this might work-and why it might be useful-a team at MIT’s Media Lab, led by physicist Joseph Jacobson and biomedical engineer Shuguang Zhang, affixed gold particles, each only 1.4 nanometers in diameter, to a piece of DNA. Each gold particle served as a tiny antenna. The researchers then exposed the DNA to radio frequency magnetic fields, causing the particles to heat up, and the double-stranded DNA to break into two strands. When they removed the magnetic field, the strands came back together immediately. “Now we have a very powerful and useful tool that can control things at the molecular level,” says Zhang. “So far, there are no tools that can do this. To be able to control one individual molecule in a crowd of molecules is very valuable.”

That value, adds postdoc Kimberly Hamad-Schifferli, arises largely from the potential ability to turn genes on and off. To do that, the MIT researchers could attach fragments of DNA to gold particles. When added to a sample of DNA, the fragments would bind to complementary gene sequences, blocking the activity of those genes and effectively turning them off. Applying a magnetic field would then heat the gold particles, causing their attached fragments of DNA to detach, in effect turning the genes back on. Such a tool could give pharmaceutical researchers a way to simulate the effects of potential drugs, which also turn genes on and off. MIT recently licensed the technology to a biotech startup, Waltham, MA-based engeneOS.

Although remote control of DNA may sound more like a parlor trick than something your doctor might use, such experiments are demonstrating that nanoelectronics can interact with biology in powerful ways. Materials like nanowires and nanotubes, extensively researched by physicists and chemists in recent years, are now in the hands of biomedical engineers like MIT’s Zhang-with huge implications for everything from drug discovery to diagnosis of diseases like prostate cancer. While it’s difficult to predict winners among these many technologies, Berkeley’s Alivisatos, for one, says, “I think these things are all going to find competitive niches.”

Fast, cheap microelectronics revolutionized the world of computing and information technology. Whether nanoelectronics can revolutionize medicine remains uncertain. But the gap between electronics and biology is fast closing, and biomedical researchers and even physicians will soon have tools to probe life’s basic molecules in ways that seemed like fantasy just a few years ago.

Sensing Success
Some companies in nanobiotech

Company	Technology Source	Strategy
Agilent Technologies (Palo Alto, CA)	Harvard University	Materials with nano-sized pores for analyzing DNA
engeneOS (Waltham, MA)	MIT	Gold nanoparticles for remote control of biological molecules
Molecular Nanosystems (Palo Alto, CA)	Stanford University	Carbon nanotubes for sensing biological molecules
Nanofluidics (Ithaca, NY)	Cornell University	Chips with nanoscale channels for analyzing DNA
NanoInk (Chicago, IL)	Northwestern University	Dip-pen nanolithography for designing biological molecules and structures
Nanosphere (Northbrook, IL)	Northwestern University	Electrode/gold nanoparticle detectors for sensing DNA and pathogens
Nanosys (Palo Alto, CA)	Harvard University	Nanowires for sensing biological molecules
SurroMed (Mountain View, CA)	Pennsylvania State University	Nanobarcodes for labeling biological molecules
U.S. Genomics (Woburn, MA)	U.S. Genomics	Nanocrystalline lattice for analyzing DNA