Biofunctional organic nanocrystals for quantitative detection of pathogen deoxyribonucleic acid

Waiting a day or more to get lab results back from the doctor's office soon could become a thing of a past.

Using nanotechnology, two research teams, taking different approaches, have developed diagnostic tests that can detect viruses as diverse as human papillomavirus (HPV), influenza, human immunodeficiency virus (HIV), and respiratory syncytial virus (RSV) in as little as 60 seconds.

Writing in the journal Nano Letters, a research team at the University of Georgia, led by Ralph Tripp, Ph.D., Yiping Zhao, Ph.D., and Richard Dluhy, Ph.D., describes its new technique based on surface enhanced Raman scattering (SERS). SERS works by measuring the change in frequency of a near-infrared laser as it scatters off viral DNA or RNA. This change in frequency, named the Raman shift for the scientist who discovered it in 1928, is as distinct as a fingerprint. This phenomenon is well known, but previous attempts to use Raman spectroscopy to diagnose viruses failed because the signal produced is inherently weak.

After experimenting with several different metals and methods, the investigators found that they could amplify a SERS signal from viral DNA using rows of silver nanorods deposited on a glass slide. And, like someone positioning a TV antenna to get the best reception, they tried several angles until they found that the signal is best amplified when the nanorods are arranged at an 86-degree angle.

"The enhancement factors are extraordinary," Dluhy said. "And the nice thing about this fabrication methodology is that it's very easy to implement, it's very cheap, and it's very reproducible."

Tripp said the technique is so powerful that it has the potential to detect DNA from a single virus particle and can also discern virus subtypes and those with mutations such as gene insertions and deletions. This specificity makes it valuable as a diagnostic tool, but also as a means for epidemiologists to track where viruses originate from and how they change as they move through populations.

The researchers have shown that the technique works with viruses isolated from infected cells grown in a lab, and the next step is to study its use in biological samples such as blood, feces, or nasal swabs. Tripp said preliminary results are so promising that the researchers are currently working to create an online encyclopedia of Raman shift values. With that information, a technician could readily reference a Raman shift for a particular virus to identify an unknown virus.

Presently, viruses are first diagnosed with methods that detect the antibodies a person produces in response to an infection. Tripp explained that these tests are prone to false positives because a person can still have antibodies in their system from a related infection decades ago. The tests are also prone to false negatives because some people don't produce high levels of antibodies.

Taking a different approach, a team of investigators led by Matthias Seydack, Ph.D., of the Berlin-based company 8sens.biognostic, used fluorescent organic nanocrystals to detect viral DNA amplified using polymerase chain reaction (PCR). The researchers, who published their work in the journal Analytica Chimica Acta, report that using these organic nanocrystals produces as much as a 147-fold increase in the sensitivity of standard PCR assays for HPV, the virus that causes cervical cancer.

To prepare the nanocrystals, the investigators mixed a common fluorescent dye with a water-soluble polymer for three days. The researchers then coated the nanoparticles with the molecule streptavidin, which forms a tight molecular coupling with a second molecule, biotin.

The researchers took advantage of this coupling by incorporating biotin into the DNA molecules produced during PCR amplification of HPV DNA using a well-established protocol. After completing a PCR amplification, the investigators simply added the organic nanocrystals and measured the resulting fluorescent signal, which was directly proportional to the amount of HPV DNA present in a sample.

The work using silver nanorods is detailed in a paper titled, "Rapid and sensitive detection of respiratory virus molecular signatures using a silver nanorod array SERS substrate." An abstract of this paper is available through PubMed. View abstract.

The work using organic nanocrystals is detailed in a paper titled, "Biofunctional organic nanocrystals for quantitative detection of pathogen deoxyribonucleic acid." Investigators from the Hong Kong University of Science and Technology, the City University of Hong Kong, and the National University of Singapore also participated in this study. This paper was published online in advance of print publication. An abstract of this paper is available at the journal's website. View abstract.