8/22/2022 1:59:35 PM  Jenny Applequist for HMNTL

Despite recent years’ dramatic improvements in cancer treatment, cancer remains second only to heart disease as a leading cause of death for Americans. But a new Nature Communications paper has reported exactly the kind of breakthrough that cancer patients yearn for: development of a highly sensitive new method for performing a liquid biopsy that can identify tiny numbers of individual cancer molecules.

Even better? The method requires only a drop or two of blood from a fingertip, meaning that a simple mail-in home test can take the place of today’s invasive biopsies and draining visits to phlebotomists.

University of Illinois researcher Brian Cunningham, one of the paper’s authors, explained that for several years, there’s been a focus on a “liquid biopsy” concept whereby one tries to monitor cancer by detecting tumor DNA circulating in the bloodstream. A problem is that circulating tumor DNA gets broken down into small fragments by enzymes in the blood, so that the DNA becomes undetectable.

“So the approach that we’re working on is an alternative that… shows a lot of promise,” he says. “Which is detecting another kind of molecule: microRNA.”

Like DNA, microRNA is a nucleic acid that, for tumors, contains a genomic sequence that originates as part of the genetic alterations that caused and drive the cancer. However, microRNA has a bonus feature: it comes packaged in an exosome, a little blob of material that protects the microRNA from the things in the blood that would otherwise tear it apart. A catch is that only a tiny number of tumor microRNA molecules will make it into the bloodstream.

“So it’s a challenge to have detection sensitivity that’s good enough to be able to see a small number of these very specific microRNA sequences,” says Cunningham, who is a professor and Intel Alumni Endowed Chair in ECE and Bioengineering.“And in the paper, we demonstrate the ability to do that.”

So how do they find the needle in the haystack?

“We’re using light-generating nanoparticles that are called ‘quantum dots’: particles made out of semiconductors that are very small, like five nanometers in diameter… and that’s not much bigger than the size of the molecules themselves that we’re trying to detect,” he says. “We can prepare the quantum dots with nucleic acid molecules that will match and bind with the microRNA molecule that we want to detect, and we can do that in such a way that one quantum dot equals one microRNA molecule.”

They then use a photonic crystal biosensor that amplifies the light from the quantum dots thousands of times over, making it possible to see individual quantum dot + microRNA pairs.

The use of the photonic crystal offers an additional benefit that surprised the team: it greatly suppresses the natural “blinking” of the quantum dots’ light. Quantum dots normally turn on and off at seemingly random times, and indeed are off most of the time. It turned out that the photonic crystal excites the dots such that “they spend the majority of their time on instead of off,” says Cunningham. “They still blink; they still go off sometimes. But they spend most of their time in the on state, which means they’re easier to see.”

A few years back, co-author Manish Kohli of the Huntsman Cancer Institute was part of a team that identified blood-based miR-375—the type of microRNA used in the present work—as “very specific in advanced metastatic castrate-resistant prostate cancer.” He explains, “It was found that high levels of miR-375 in the blood of [these] patients not only correlated with worse patient outcomes but also predicted that use of a common chemotherapy in advanced prostate cancer, called docetaxel, will not work.”

Kohli says that the present paper is only an early step in an ongoing campaign of ambitious research. The next step will be to figure out how to apply the new methods to advanced prostate cancer patients’ fingerstick blood samples and “what that tells us in terms of the survival of the cancer patient or outcomes of treatments using different drugs.” This work has already been started using cancer patients’ samples.

Cunningham echoes Kohli’s excitement about the work to come. “We just have a wonderful team. This type of research is very multidisciplinary, and we have an excellent team of the clinical and translational researchers at Huntsman, we have people working on new biochemistry methods to do the detection, we have sensor people working on the biosensor and detection instrument; we have Prof. Andrew Smith, who’s one of the leaders in the world on quantum dots. We have big ambitions… for extending the capabilities of this approach.” He adds that the graduate students and postdocs, led by first author Yanyu Xiong, demonstrated great ingenuity and careful attention to all the detailed methods needed to prove the new physics principles presented in the paper.

One ultimate goal will be to leverage the tests’ ease and precision to understand changes in a patient’s cancer over time, so that treatment can be adjusted accordingly. While something like a CT scan can only provide crude information—say, that a tumor shrank by 20%—a microRNA test could give clinicians more quantitative and precise information about what’s happening with the tumor, so they can make the most appropriate choices among various treatment options. The simple new testing approach should also make it far easier to monitor cancer survivors for signs of returning cancer.

“If successful, this has the potential to be the next generation of liquid biopsies for cancer patients,” concludes Kohli.

The paper is “Photonic crystal enhanced fluorescence emission and blinking suppression for single quantum dot digital resolution biosensing” by Yanyu Xiong, Qinglan Huang, Taylor D. Canady, Priyash Barya, Shengyan Liu, Opeyemi H. Arogundade, Caitlin M. Race, Congnyu Che, Xiaojing Wang, Lifeng Zhou, Xing Wang, Manish Kohli, Andrew M. Smith & Brian T. Cunningham, Nature Communications, vol. 13 (2022), https://doi.org/10.1038/s41467-022-32387-w.

The project is part of the Center for Genomic Diagnostics, which is a joint activity of the Holonyak Micro & Nanotechnology Lab and the Carl R. Woese Institute for Genomic Biology.

New label-free detection technique digitally counts intact SARS-CoV-2 virus particles in saliva or exhaled breath

As health and research institutions continue to rapidly develop new methodologies for detecting SARS-CoV-2, researchers from the Holonyak Micro & Nanotechnology Laboratory have found themselves at both forefronts of discovery and featured on the cover of the Journal of the American Chemical Society with their paper: Label-free Digital Detection of Intact Virions by Enhanced Scattering Microscopy.

Label-free detection, an approach that utilizes a biosensor and detection instrument for viral load monitoring, is a solution that can capture and digitally count intact virus particles in saliva or exhaled breath to provide lower cost and reduced time to diagnose an infection.

Currently, the most widely used SARS-CoV-2 PCR testing method is the PCR assay, which uses enzymatic amplification to make many copies of a specific section of the virus’s RNA, which requires extraction of the viral genome and a complex laboratory procedure.  Instead, the new approach uses a specially designed nucleic acid molecule, called an “aptamer” attached to a biosensor that selectively recognizes one of the proteins on the virus outer surface, and captures it in a single step at room temperature, with no other reagents required.  Once captured, the viruses are counted, using a newly invented form of microscopy, that generates images from laser light that scatters from each captured virus.

“Our technique requires only the saliva sample, and avoids the need for any additional reagents, thus we expect the cost for a test to be significantly reduced and the overall process to be greatly simplified (less labor-intensive),” said co-author Nantao Li, a graduate student in electrical and computer engineering. “In addition, the aptamers we used can selectively differentiate between active viruses from inactive ones, thus providing more robustness for diagnosis results. Conventional techniques, such as PCR, detect the viral RNA sequence which can remain in bodily fluids even after infectious viruses are no longer present.”

To detect and count the captured viruses, the team recently invented a new imaging approach called Photonic Resonator Interferometric Microscopy (PRISM). PRISM uses a photonic crystal biosensor surface to enhance light scattering from virus particles. The photonic crystal is a nanostructured surface that provides two effects. First, it enables each virus to scatter more light from an illuminating laser, which increases their signal contrast.  Secondly, the photonic crystal directs the scattered light toward the microscope objective – allowing a larger fraction of the scattered light to be collected.

While label-free digital detection can be a promising alternative to traditional SARS-CoV-2 detection, principal investigator Brian Cunningham believes this approach can be widely applicable to other areas.

“We are already making plans soon to perform viral load monitoring of HIV in plasma, and we are building a more portable version of the detection system that would be small and inexpensive enough to perform well in biology labs or diagnostic lab facilities,” said Cunningham, Intel Alumni Endowed Chair in Electrical and Computer Engineering.

Moving forward, the research team – comprised of principal investigators Brian Cunningham, Yi Lu, Xing Wang, and co-authors Xioajing Wang, Nantao Li, and Joseph Tibbs, are designing and implementing a new type of capture molecule that will reduce the detection time to a few minutes. They envision the future capability for a person to exhale into a device, and for exhaled virus particles to be captured on the sensor.

To read more about Label-free Digital Detection of Intact Virions by Enhanced Scattering Microscopy, you can find it published in the Journal of American Chemical Society here. 

CUNNINGHAM GROUP DEVELOPS MICROSCOPE TECHNIQUE TO DETECT INDIVIDUAL VIRUSES FOR POWER RAPID DIAGNOSTICS

CHAMPAIGN, Ill. — A fast, low-cost technique to see and count viruses or proteins from a sample in real time, without any chemicals or dyes, could underpin a new class of devices for rapid diagnostics and viral load monitoring, including HIV and the virus that causes COVID-19.

Researchers at the University of Illinois Urbana-Champaign described the technique, called Photonic Resonator Interferometric Scattering Microscopy, or PRISM, in the journal Nature Communications.

“We have developed a new form of microscopy that amplifies the interaction between light and biological materials. We can use it for very rapid and sensitive forms of diagnostic testing, and also as a very powerful tool for understanding biological processes at the scale of individual items, like counting individual proteins or recording individual protein interactions,” said Illinois ECE Professor and study leader Brian T Cunningham, the Intel Alumni Endowed Chair of electrical and computer engineering and a member of the Holonyak Micro and Nanotechnology Lab and the Carl R. Woese Institute for Genomic Biology at Illinois.

In optical microscopes, light bounces off any molecules or viruses it encounters on a slide, creating a signal. Instead of a regular glass slide, the PRISM technique uses photonic crystal: a nanostructured glass surface that brilliantly reflects only one wavelength of light. Cunningham’s group designed and fabricated a photonic crystal that reflects red light, so that the light from a red laser would be amplified.

“The molecules we are looking at – in this study, viruses and small proteins – are extremely small. They cannot scatter enough light to create a signal that can be detected by a conventional optical microscope,” said graduate student Nantao Li, the first author of the paper. “The benefit of using the photonic crystal is that it amplifies the light’s intensity so it’s easier to detect those signals and enables us to study these proteins and viruses without any chemical labels or dyes that might modify their natural state or hinder their activity – we can just use the intrinsic scattering signal as the gauge for determining if those molecules are present.”

The researchers verified their technique by detecting the virus that causes COVID-19. PRISM detected individual coronaviruses as they traveled across the slide’s surface. The researchers also used PRISM to detect individual proteins such as ferritin and fibrinogen. The technique could allow researchers to study such biological targets in their natural states – watching as proteins interact, for example – or researchers could seed the surface of the photonic crystal slide with antibodies or other molecules to capture the targeted items and hold them in place.

“It takes 10 seconds to get a measurement, and in that time we can count the number of viruses captured on the sensor,” Cunningham said. “It’s a single-step detection method that works at room temperature. It is also fast, very sensitive and low cost. It’s very different from the standard way we do viral testing now, which involves breaking open the viruses, extracting their genetic material and putting it through a chemical amplification process so we can detect it. That method, called PCR, is accurate and sensitive, but it requires time, specialized equipment and trained technicians.”

Cunningham’s group is working to incorporate PRISM technology into portable, rapid diagnostic devices for COVID-19 and HIV viral load monitoring. The group is exploring prototype devices that incorporate filters for blood samples and even condensation chambers for breath tests.

“We are also going to use this as a research tool for biology and cancer,” Cunningham said. “We can use it to understand protein interactions that are parts of disease processes. We are interested in using it to detect these tiny vesicles that cancer cells shed, and to see what tissues they come from, for diagnosis, and also to study what cargo they are transporting from the cancer cells.”

The National Science Foundation and the National Institutes of Health supported this work. Cunningham is affiliated with the Beckman Institute and HMNTL.