Label-Free Nanosensing Technique Helps Detect Early Biomarkers of Cancer

A new technique developed by biomedical engineers from Duke University can be used to concurrently identify the presence of various specific microRNAs in RNA derived from tissue specimens and without the necessity for target amplification or labeling.

Through this latest technique, early biomarkers of cancer and other disorders could be detected without the need for costly, extensive, and time-intensive procedures as well as exclusive laboratory instruments demanded by present-day technologies.

The study results were published online in the Analyst journal on May 4^th, 2020.

The general research focus in my lab has been on the early detection of diseases in people before they even know they’re sick. And to do that, you need to be able to go upstream, at the genomic level, to look at biomarkers like microRNA.

Tuan Vo-Dinh, R. Eugene and Susie E. Goodson Distinguished Professor, Biomedical Engineering, Duke University

Vo-Dinh is also the director of the Fitzpatrick Institute for Photonics.

MicroRNAs are essentially short RNA molecules that adhere to messenger RNAs and prevent them from sending their instructions to the protein-producing machines of the body. This process could effectively control the expression of genes or silence specific parts of DNA, thus modifying the behaviors of particular biological functions.

So far, researchers have identified over 2000 microRNAs in humans that influence metabolism, differentiation, growth, and development.

Thanks to discoveries and a better understanding of these small genetic packages, several microRNAs have been implicated in the misregulation of biological functions, leading to various medical disorders, spanning from Alzheimer’s disease to brain tumors. Such discoveries have resulted in a growing interest in applying microRNAs as therapeutic targets and disease biomarkers.

Since only trace amounts of miRNAs are present in bodily samples, conventional techniques of analyzing these miRNAs involve genetic-amplification procedures like RNA sequencing and quantitative reverse transcription PCR (qRT-PCR).

Although such technologies are known to perform quite well in research studies and in well-equipped laboratories that can take months or even years, they are not ideally suitable for rapid diagnostic results either out in the field or at the clinic. Therefore, to close this gap in terms of applicability, Vo-Dinh and his collaborators are using silver-plated gold nanostars.

Gold nanostars have multiple spikes that can act as lighting rods for enhancing electromagnetic waves, which is a unique feature of the particle’s shape. Our tiny nanosensors, called ‘inverse molecular sentinels’, take advantage of this ability to create clear signals of the presence of multiple microRNAs.

Tuan Vo-Dinh, R. Eugene and Susie E. Goodson Distinguished Professor, Biomedical Engineering, Duke University

Vo-Dinh also holds a faculty appointment in Duke Chemistry.

Although the term is definitely a mouthful, the fundamental concept with regard to the design of the nanosensor is to cause a label molecule to shift extremely close to the spikes of the star when a particular stretch of target RNA is detected and trapped.

When the triggered sensor is illuminated with a laser, the label molecule shines very brightly due to the lightning rod effect of the nanostar tips. This indicates that the target RNA has been captured.

To set this trigger, the scientists tethered a label molecule to one of the points of the nanostar using a stretch of DNA. While the DNA is designed to curl in on itself in a loop, it is kept open by an RNA “spacer” that is customized to adhere to the target microRNA being tested.

When that specific microRNA comes by, it binds to the spacer and removes it, enabling the DNA to curl in on itself in a loop fashion and bring the label molecule almost close to the nanostar.

When the label molecule is excited by laser, it produces a light known as a Raman signal, which is usually quite weak. However, the shape of the nanostars—combined with a coupling effect of individual reactions induced by the silver coating and gold nanostars—intensifies the Raman signals by several million times, rendering them easier to identify.

The Raman signals of label molecules exhibit sharp peaks with very specific colors like spectral fingerprints that make them easily distinguished from one another when detected. Thus we can actually design different sensors for different microRNAs on nanostars, each with label molecules exhibiting their own specific spectral fingerprints. And because the signal is so strong, we can detect each one of these fingerprints independently of each other.

Tuan Vo-Dinh, R. Eugene and Susie E. Goodson Distinguished Professor, Biomedical Engineering, Duke University

For this clinical research, Vo-Dinh and his research group teamed up with Katherine Garman, associate professor of medicine, and collaborators from the Duke Cancer Institute to apply the novel nanosensor platform. The aim was to prove that nanosensors are capable of detecting miR-2—a certain microRNA linked to very early phases of esophageal cancer—equally well as other more extensive sophisticated techniques.

In this example, the use of miR-21 is more than sufficient to differentiate cancerous samples from healthy tissue samples. But for other disorders, several other microRNAs may need to be detected to obtain a consistent diagnosis, which is actually why the scientists are thrilled by the common applicability of their inverse molecular sentinel nanobiosensors.

“Usually three or four genetic biomarkers might be sufficient to get a good diagnosis, and these types of biomarkers can unmistakably identify each disease,” added Vo-Dinh. “That’s why we’re encouraged by just how strong of a signal our nanostars create without the need of time-consuming target amplification.”

Vo-Dinh continued, “Our method could provide a diagnostic alternative to histopathology and PCR, thus simplifying the testing process for cancer diagnostics.”

To patent his new nanostar-based biosensors, Vo-Dinh has been working with his collaborators and Duke University’s Office of Licensing and Ventures for over three years. With the recently awarded patent, the scientists are looking forward to test the limitations of their technology’s capacities and explore the possibilities of technology transfer with the private industry.

“Following these encouraging results, we are now very excited to apply this technology to detect colon cancer directly from blood samples in a new NIH-funded project. It’s very challenging to detect early biomarkers of cancer directly in the blood before a tumor even forms, but we have high hopes,” concluded Vo-Dinh.

The National Science Foundation (1106401) and National Institutes of Health (1R21CA196426) have funded the study.

Journal Reference:

Crawford, B. M., et al. (2020) Plasmonic nanobiosensors for detection of microRNA cancer biomarkers in clinical samples. Analyst. doi.org/10.1039/D0AN00193G.