Nanotech Group's "Bio-Barcode" Test a Promising Diagnostic, Research Tool

When Chad Mirkin, PhD, envisions nanotechnology's future, he scoffs at the notion that it will create such sci-fi wonders as microscopic robots. But he wholeheartedly believes it will have a dramatic impact on the practice of medicine.

Sitting back in his spacious office in shorts and a casual shirt, Mirkin, who heads up Northwestern University's Institute for Nanotechnology, in Evanston, Ill, recently talked with JAMA about an innovative nanotech detection tool for diagnostic tests and clinical research that he and colleagues have developed, the bio-barcode assay.

"It could completely change the whole landscape in terms of medical diagnostics," says Mirkin, a 40-year-old dynamo who has an array of prestigious awards and patents and more than 200 publications to his credit. "Has it changed it yet? No. But this is a new analytical tool that could change the way doctors think."

At the Institute, the first such federally funded nanotechnology institute in the country, Mirkin and a cohort of graduate students and postdocs have spent the past decade developing the assay. The tool is similar to today's polymerase chain reaction (PCR), the revolutionary technique that allows the detection and amplification of tiny amounts of DNA. But it has a number of advantages over PCR.

Compared with PCR, which is limited to DNA, "the bio-barcode assay works for DNA, RNA, proteins, and certain small molecules and metal ions," explains Mirkin. "That is extraordinarily general."

Mirkin's group has demonstrated the assay's effectiveness to detect minuscule levels of prostate-specific antigen (PSA) (Science. 2003;301:1884-1886) and anthrax DNA (J Am Chem Soc. 2004;126:5932-5933). The bio-barcode assay detected as few as 18 to 20 PSA molecules in 10 µL of solution, a level of detection that is 6 orders of magnitude more sensitive than conventional clinical assays. And 10 copies of anthrax DNA were detected in 30 µL of solution, a sensitivity comparable to PCR-based techniques.

The new assay has many qualities that could make it a more widespread clinical tool. "We've got simplicity, we've got speed, we've got sensitivity, we've got low cost," he says of the bio-barcode assay. The group also has developed a point-of-care prototype that is half the size of a credit card. Therefore, physicians could even carry this self-contained apparatus in their pockets.

Part of the technology's simplicity stems from its ability to detect targets without the use of enzymes, which PCR and many other techniques require. "So you don't have to deal with the complexity associated with enzymes or a lot of the contamination or sample storage issues of enzymes," he notes.

The sensitivity of Mirkin's assay could allow physicians to detect seemingly infinitesimal amounts of target DNA or protein in tissue or blood samples. The ability to detect such low concentrations of biologically relevant molecules could allow scientists to "begin improving existing tests that benefit from higher sensitivity or . . . to think about markers that one couldn't even think about with old technology," says Mirkin.

A particularly promising application for the technique may be early HIV detection or assessment of patients taking antiretroviral drugs that suppress viral replication to levels below the level of detection by conventional tests. Small amounts of HIV proteins and genetic material are present in such patients, and being able to detect a slight increase could give physicians an early heads-up if treatment begins to fail.

"We've used [the bio-barcode assay] for detecting HIV directly in clinical samples and have been able to achieve levels of detection for HIV proteins and RNA that surpassed the current technologies," says collaborator Steven Wolinsky, MD, of Northwestern Memorial Hospital, in Chicago. He is also studying the assay's ability to detect the single point mutations in HIV that confer antiretroviral resistance.

The technique holds promise for a wide range of clinical applications, including diagnosis at point of care, blood banking applications, detection of point mutations in screening for inborn errors of metabolism, and prenatal screening for disorders, says Wolinsky. "It really is a quantum leap."

Mirkin is creating panels that can simultaneously look at different Alzheimer disease markers, with the hope of helping clinicians diagnose the disease. Right now, however, researchers disagree on which markers are clear indicators of the disease, but the assay's ability to detect low concentrations of biologically relevant molecules may help resolve this issue, Mirkin suggests.

"They can't even begin to make the link [between a marker and the disease] because they can't detect these markers with conventional assays because the target concentration is so low," he explains.

William Klein, PhD, also of Northwestern, recently found that small, soluble molecules of amyloid β-peptide may cause memory loss in patients with Alzheimer disease (Proc Natl Acad Sci U S A. 2003;100:10417-10422). This peptide is found at elevated levels in the brains of patients with Alzheimer disease, and it can diffuse into the blood at low concentrations.

"In our pilot experiments in our collaborations with Dr Mirkin's group, it's been possible to detect as few as 50 molecules [of amyloid β-peptide]," says Klein. "This technology has an ultrasensitive ability to detect either DNA or disease-relevant proteins," he added.

Another Mirkin collaborator at Northwestern, Lester Binder, PhD, studies tau, a major protein component of abnormal tangles found in brain cells of patients with Alzheimer disease.

"People have always thought that if you had a way to test for evidence of abnormal tau in cerebral spinal fluid or blood of patients, you would have a premortem way to diagnose Alzheimer disease," says Binder. A sensitive tau detection test could also be used to study the progression of the disease and its response to experimental drugs, he suggests. Mirkin is working with Binder to use the assay to detect a cleaved form of tau that is a strong indicator that nerve cells are undergoing apoptosis, or programmed cell death.

The bread box-sized apparatus that Mirkin uses to run bio-barcode assays in his laboratory looks simple enough, but the reactions that occur within it are quite complex.

The DNA detection technique for anthrax DNA, for example, requires two types of tiny spherical probes: a magnetic particle, 1 micrometer in diameter, and a gold nanoparticle, 13 nanometers in diameter. Tethered to each of these probes are single-stranded DNA molecules that are complementary to distinct segments of anthrax DNA.

The gold nanoparticle probe is also tethered to hundreds of "barcode" DNA sequences, which play a role analogous to the barcodes on supermarket items. Just as a barcode on a box of cornflakes is simply a tagging mechanism designed to be detected when a clerk swipes the box across the checkout scanner, the barcode DNAs in Mirkin's technique are present solely to serve as tags that the assay's "scanner"—a gene chip—can recognize.

Detecting anthrax DNA (or other targets of choice) involves 5 basic steps (as illustrated in the figure on page 1292). First, barcode-binding DNA sequences and DNA complementary to anthrax DNA are attached to the the appropriate probes; barcode DNA is then bound to the barcode-binding DNA. Second, the anthrax-seeking probes are exposed to a sample. If the sample contains anthrax DNA, this DNA binds to and becomes sandwiched between the two probes. In step 3, a magnetic field is applied that gathers up all of the magnetic microparticle probes—including those that are bound to anthrax DNA plus the barcoded gold nanoparticle probes. The barcode DNA fragments are then released from the gold nanoparticle in step 4, and detected and analyzed on a gene chip in step 5, revealing the presence of anthrax DNA. Because the barcode-studded gold probes get dragged along during the magnetic field step only if anthrax DNA is present to bridge the two probes, the absence of barcode DNA in the final step translates to a negative result, indicating no anthrax is present.

The selectivity of this approach for detecting DNA sequences is so impressive that a gene with a single nucleotide mutation can be distinguished from the normal version. This makes it attractive for diagnosing some diseases as well as analyzing DNA variations called single-nucleotide polymorphisms.

It can also be used to screen for many different DNA sequences at once, because different barcode fragments can be assigned to different targets. "The beauty of this is that . . . I can, in one sample, add 20 different barcode probes; if the targets are around, all of them will appear as positives in the assay readout," says Mirkin.

The protein detection technique uses barcode DNA as well, but antibodies against the target protein are tethered to the probes instead of complementary DNA. In the protein assay, the antibodies are responsible for sandwiching the protein target of interest, while the barcodes again serve as a tag for a given protein, and are responsible for reporting its presence.

Mirkin predicts that nanotechnology could soon play major roles in clinical diagnosis and treatment. A diagnostic system called the Verigene ID has been commercialized by Nanosphere Inc, a Northbrook, Ill, company cofounded by Mirkin. It is being tested in hospitals around the country and should reach the market next year.

Verigene ID incorporates Mirkin's nanoparticle technologies and is designed to enable physicians to detect patient predispositions to diseases such as cancer and nervous system disorders and optimize patient drug response based on genetic variations. It is also in development as a field-deployable system that tests for such hazardous biological weapons as ricin and botulinum toxin.

The company is tapping into other techniques coming out of Mirkin's laboratory as well. "There's a plethora of patents he's produced that we're using," says Nanosphere's Chief Technology Officer, Bill Cork. "It's kind of like trying to drink from a fire hose, because he's come up with a lot of ways to detect things using nanoparticles."