Direct Detection of Intact SARS-CoV-2 with PCR Sensitivity

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. 

New Point of Care Diagnostic Instrument for Cancer Biomarkers

October 18, 2021
By: Ananya Sen

Current medical diagnostics involve sending samples to laboratory facilities, which can be difficult and expensive. Researchers at the University of Illinois Urbana-Champaign have designed a desk-sized instrument that can make the same measurements at the location where the samples are collected.

For several years the Cunningham group has been developing microscopes that use photonic crystal biosensors—nanostructured glass surfaces that brightly reflect only one wavelength of light. “Although our original photonic crystal microscope is very versatile, it’s the size of a ping pong table,” said Brian Cunningham (CGD Director/MMG), the Intel Alumni Endowed Chair of Electrical and Computer Engineering. “We wanted to build a portable instrument that had the same detection capabilities. The new one we built can easily fit on a desk and costs around $7,000, compared to the non-portable microscope, which costs $200,000.”

The researchers had previously developed the larger photonic crystal microscope so that it could provide a strong contrast counting surface-attached gold nanoparticles, a feature that the portable version also shares. “The photonic crystals act like a mirror, but only for the color red. The gold nanoparticles are non-reflective and show up as dark spots,” Cunningham said. The microscopes can, therefore, be used to detect proteins or other biomarkers that are linked to the gold nanoparticles.

The portable versions use a red LED light, which gets reflected off the photonic crystal, and the image is captured by a webcam. “The system itself is quite easy to build and can be put together within a day,” said Nantao Li, a graduate student in the Cunningham lab and co-first author on the paper.

The portable microscope was used to detect specific microRNAs—small, single-stranded, non-coding RNAs—that are associated with prostrate cancer. Each gold nanoparticle was attached to a single-stranded piece of DNA, called the probe, which in turn was attached to pieces of single-stranded protector DNA. If the target miRNA sequence was present in the sample, it would displace the protector DNA, which would cause the nanoparticle to bind to the photonic crystal, according to Shreya Ghosh, a previous postdoctoral researcher in the Cunningham lab and the other co-first author of the paper.

Since almost every cancer has miRNAs associated with it, the microscope can, in theory, be used to detect different cancer types. “We are collaborating with researchers at the Mount Sinai Medical Center to diagnose lung cancer, and with Huntsman Cancer Institute to measure the effects of chemotherapy in prostate cancer,” Cunningham said. “We will also be working with Carle Hospital to detect miRNAs found in the blood of breast cancer patients.”

The researchers are working to lower the microscope cost even further. “We are trying to use smartphone cameras to capture the images. Our aim is to reduce the cost to less than $100,” Li said. They are also trying to build handheld versions of the microscope, which would consist of a box with an image sensor that can communicate wirelessly with a smartphone.

The Cunningham lab is currently seeking to establish a Center for Enhanced Biosensor Microscopy at the IGB, where they will be able to train researchers throughout the academic research community to use these instruments to detect any type of biomarker.

The study “A compact photonic resonator absorption microscope for point of care digital resolution nucleic acid molecular diagnostics” was published in Biomedical Optics Express. The work was funded by the National Institutes of Health, Jump ARCHES endowment through the Health Care Engineering Systems Center, Zhejiang University ZJU-UIUC Joint Research Center, Ronald H. Filler Scholarship for Cancer Scholars, and the Illinois Scholars in Undergraduate Research Scholarship.

Introducing PRISM: Photonic Resonance Interferometric Scattering Microscopy

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.”

PRISM for COVID-19 detection. At top, concept art. Bottom left, a microscope image of a single virus on the photonic crystal surface. Bottom right, a PRISM image with six viruses detected. Image courtesy of Nantao Li
PRISM for COVID-19 detection. At top, concept art. Bottom left, a microscope image of a single virus on the photonic crystal surface. Bottom right, a PRISM image with six viruses detected.
Image courtesy of Nantao Li

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.

 

Establishing the Center for Pathogen Diagnostics

9/30/2020, Kim Gudeman

With COVID-19 infecting more than 25.1 million worldwide to date, the pandemic has underscored the need for cost-effective, accurate, and quick diagnostics.

The University of Illinois, Urbana-Champaign and Zhejiang University are launching the Center for Pathogen Diagnostics (CPD) to create new detection systems that address limitations of current technologies, while leveraging the power of artificial intelligence and machine learning to analyze disease trends, analyze sensor data, and predict future outbreaks. The new devices – which will range from wearable sensors to mobile point-of-care devices to large laboratory instruments – will be able to detect pathogens that cause viral, bacterial, fungal, environmental, and food-borne illnesses.

“With COVID, we’re seeing the limitations of current best practices for virus detection,” said Brian Cunningham, the Intel Alumni Endowed Chair in the Department of Electrical and Computer Engineering at Illinois. “It takes too long to get results, and accuracy remains a concern. Instead of chemical testing, we’re interested in using new detection modalities, like detecting intact viruses, to create faster, more cost-effective diagnostics.”

The team, which is comprised of researchers at Illinois and ZJU, represents five “pillars” of pathogen diagnostic technologies. First, researchers will study the interactions between pathogens and host cells at the molecular level, which will provide targets for new pathogen detection platforms.

Second, the team will develop sample pre-processing techniques that enable the breakdown of cells and extraction of DNA and RNA, so that viruses and bacteria can be identified. This step will help researchers create a device capable, for example, of differentiating COVID-19 (SARS-CoV-2) from influenza, which, while both viruses, have features that can be used to distinguish them from each other.

Researchers also will create biosensors that are more sensitive, which will lower false negatives and positives, and be able to detect different variations of SARS-CoV-2. One important direction is development of mobile smartphone-based platforms that allow test results to be obtained immediately after gathering a sample, and the fabrication of microfluidic devices that automate the pre-processing of test samples before the detection step.

Finally, the team will use AI and machine learning algorithms to analyze data that is produced by the sensors, and to monitor health trends for public health officials. For example, AI could be used to provide better modeling for disease transmission or to reduce the number of physical tests needed in a community by making inferences about infection rates from a small subset of the population.

Transitioning the technology to the marketplace will be an important component of the center as well, says Cunningham.

“The purpose of the center is to do innovative research that actively improves global public health,” he said. “Commercialization is one of the outcomes that we’re working towards.”

The Center for Pathogen Diagnostics will be located in the Holonyak Micro & Nanotechnology Laboratory. In addition to Cunningham, the Illinois research team includes: Yang Zhao, HMNTL Director Xiuling Li, and Lav Varshney, electrical and computer engineering; Yi Lu, chemistry; Steven Blanke, microbiology; and Xing Wang, Holonyak Micro & Nanotechnology Lab. The ZJU team includes Shaowei Fang, Qingjun Liu, Chun Zhou, Huan Hu, and Yu Lin.

The new five-year, $1.5 million center is not the first collaboration between the two universities. In 2016, The Zhejiang University-University of Illinois at Urbana-Champaign Institute (ZJUI) was launched on ZJU’s international campus in Haining, China, about 120 km southwest of Shanghai. ZJU-UIUC Institute faculty teach and research in broad program themes of engineering and system sciences; information and data sciences; and energy, environment, and infrastructure sciences. The Center is one of three research efforts that was just funded through the ZJUI initiative.

“The Center for Pathogen Diagnostics is another great example of the fruitful partnership that Illinois enjoys with ZJUI,” said Dean Rashid Bashir of The Grainger College of Engineering. “We look forward to working with ZJUI on solving urgent problems that are relevant to the entire global community.”

Portable Smartphone COVID Test

As COVID-19 continues to spread, bottlenecks in supplies and laboratory personnel have led to long waiting times for results in some areas. In a new study, University of Illinois, Urbana-Champaign researchers have demonstrated a prototype of a rapid COVID-19 molecular test and a simple-to-use, portable instrument for reading the results with a smartphone in 30 minutes, which could enable point-of-care diagnosis without needing to send samples to a lab.

The paper can be found on the Nanosensors Group “Publications” page, and at the Proceedings of the National Academy of Sciences website:

https://www.pnas.org/content/early/2020/08/27/2014739117

The press release to this news story can be found at:

https://news.illinois.edu/view/6367/241622493

Using Photonics to Generate “Hot Electrons” that Catalyze Chemical Transformations

Researchers in Prof. Brian Cunningham’s Nanosensors Group at the University of Illinois, in collaboration with Prof. Singamaneni’s research group at Washington University, described a new approach for efficiently catalyzing chemical reactions using light, in the journal ACS Photonics.   The researchers harnessed a new approach for amplifying electromagnetic fields in nanometer-scale volumes by coupling the energy from a laser into a nanostructured photonic crystal surface.  The electromagnetic fields in the photonic crystal resonate with the laser’s wavelength, and when a metal nanoparticle is placed onto the photonic crystal, the electrons in the metal resonate as well.    A portion of the resonating electrons become more reactive than ordinary electrons, and are able to transfer to nearby chemical molecules, and thus catalyze specific chemical reactions.  The reactive electrons are often referred to as being “hot,” even though they are not hot in the temperature sense. The research shows that, by coupling  laser light to a photonic crystal, chemical reactions are driven forward with greater efficiency, allowing less energy to perform a process than possible without the photonic crystal.  Because the approach uses low illumination power that can be distributed over large surface areas, we envision the potential for optically driven chemical reactors.  The reactors would be only several micrometers in height, with transparent windows, an inlet for precursors, an outlet for products, and nanoparticle-coated photonic crystals comprising the upper and lower surfaces.

ECE graduate student Qinglan Huang and IGB Fellow Taylor Canady led the research, which was performed in the Holonyak Micro and Nanotechnology Laboratory.  The paper, entitled “Enhanced Plasmonic Photocatalysis through Synergistic Plasmonic–Photonic Hybridization“, by  Q. Huang, T.D. Canady, G. Gupta, N. Li, S. Singamaneni and B.T. Cunningham, can be found at:  ACS Photonics (2020).

Check out the final paper here: FINAL published ACS2020.

Digital Diagnostics Review Paper in Lab on a Chip

The Nanosensors Group from the University of Illinois at Urbana-Champaign published a Critical Review for the journal Lab on a Chip, entitled “Digital Resolution Biomolecular Sensing for Diagnostics and Life Science Research.”  The co-authors of the paper are Qinglan Huang, Nantao Li, Hanyuan Zhang, Congyu Che, Fu Sun, Yanyu Zhang, Taylor Canady,  and Brian Cunningham.  The paper reviews an important and very challenging frontier of the field of biosensing, where novel technologies are demonstrating the ability to digitally count molecules with single-unit precision. Through the use of enzymatic chemical reactions, nanoparticle tags, ultra-sensitive transducers, multiplexing detection instruments, and novel biochemistry, the reviewed technologies are finding applications in life science research, drug discovery, and diagnostics.

The paper can be found on the Nanosensors Group “Publications” page, and at the Lab on a Chip web site:

https://pubs.rsc.org/en/content/articlelanding/2020/lc/d0lc00506a#!divAbstract

 

 

Inexpensive, portable detector identifies pathogens in minutes

Editor’s notes:

To reach Brian Cunningham, call 217- 265-6291; email bcunning@illinois.edu.

The paper “Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract” is available online and from the U. of I. News Bureau. DOI: 10.1039/D0LC00304B

Interview on WILL about COVID Diagnostics

U OF I RESEARCHERS DEVELOPING SMARTPHONE APP FOR CORONAVIRUS TESTING

A team of University of Illinois researchers is working on a smartphone application that would detect the novel coronavirus within 30 minutes, without the need for a diagnostic lab.

Illinois Newsroom’s Brian Moline spoke with Brian Cunningham. He’s a professor of electrical and computer engineering, and a professor at Nick Holonyak Jr., Micro and Nanotechnology Lab on the Urbana campus.

He said that’s just one of several diagnostic technologies that his research team is working on in collaboration with others.

“One test we’re developing is based on detecting the nucleic acid sequence, part of the genome of the virus,” Cunningham said. “In that test, you take the virus, break it apart by putting it into a chemical that breaks the virus membrane and releases the RNA material. And then, in that test, you take that genomic sequence, you have a special molecule that recognizes it, and then turns each individual piece of the genome into thousands and then millions of individual copies that fluoresce. That test is performed inside a small, plastic cartridge that has an output that is readable by a phone.”

Cunningham said there are both advantages and disadvantages in working with smartphones.

“Of course, now, everybody has one,” he said. “It’s a powerful device that you can use in many ways, but you can also use it in measuring biological diagnostic tests. The downside is that it’s not approved by regulatory agencies to use a mobile device like that, for doing a test, and you have to show very rigorously that a phone-based system can give results that are equivalent to laboratory tests using clinical samples.”

Cunningham said his group has completed successful testing of the smartphone application using respiratory viruses from horses. He has submitted the research to the journal Lab on a Chip to be considered for fast-track publication. You can read that submission below.

Cunningham said they have also submitted applications for emergency funding to the National Science Foundation and the National Institutes of Health.

In addition to the smartphone application, Cunningham said his research team is also working on a biosensor method of testing that detects and counts the intact coronavirus from nasal swab extract. He said they are also working on another test that would detect if a person has antibodies that could give them immunity to COVID-19.

Announcing the Center for Genomic Diagnostics

January 29, 2020
By: Claudia Lutz

A new research center at the University of Illinois directed by Donald Biggar Willett Professor in Engineering Brian Cunningham aims to revolutionize diagnostics and personalized medicine, developing technologies that are at once more accurate, more affordable, and more practical for routine care.

The Carl R. Woese Institute for Genomic Biology (IGB) and the Grainger College of Engineering are working together to support the launch the new Center for Genomic Diagnostics (CGD), which will be housed within the IGB. The CGD will also take advantage of specialized laboratory space and equipment in Illinois’ Holonyak Micro and Nanotechnology Lab.

The vision for the new center begins with molecules called biomarkers that are naturally produced as part of a healthy biological state or disease process. If such a molecule is produced in detectably larger or smaller quantities in certain conditions, it can serve as the basis for a reliable test for that condition. For example, the hormone human chorionic gonadotropin, which is detected in urine by home tests during early pregnancy, is a biomarker. Other biomarkers signal the presence of diseases, including some types of cancer.

“Our goal for the center is first to use genomics and bioinformatics to identify novel biomarkers,” Cunningham said. “As we seek to validate how biomarker presence and concentration changes with a specific health condition, we’re also interested in developing novel biochemistry methods for selectively detecting those molecules with methods that are simple, yet extremely sensitive.”

The concept for the center emerged from Cunningham’s research theme at the IGB, Omics Nanotechnology for Cancer Precision Medicine, which was established in 2016. The theme brings together computational, biochemical, biomedical, and engineering approaches to biomarker discovery and innovations in the realm of biomarker detection. In its conversion from a theme to a center, members are broadening their focus on cancer to include a wide variety of diseases and conditions.

“There are biomarkers for diseases other than cancer, as well as biomarkers that provide information on a person’s nutrition, environment, microbiome, and metabolism,” Cunningham said. “There are genomic biomarkers for infection and immunity, for inflammation and sepsis, and surprisingly even for environmental exposure, psychological stress, and anxiety . . . So that’s how we’re turning from a cancer group into a genomic diagnostic center, so we can consider things more broadly.”

The center already works closely with the Cancer Center at Illinois. With its expanded scope, the CGD is also growing its relationships with Illinois’ Health Care Engineering Systems Center, as well as the Mayo Clinic and Illinois Alliance.

“Our diagnostic [capability] is one of the things that allows medicine to be personalized,” Cunningham said. “So if you can have a test for a biomarker that tells the clinician something about a particular patient, they have a specific gene is being expressed, or they have a protein molecule that is present in high levels, the information can indicate that the patient is more likely to have successful treatment with a particular drug.”

The faculty and research staff that will join the center from the original IGB research theme and from around campus represent a diversity of backgrounds and expertise.

“Our team includes faculty with backgrounds that span bioinformatics, biochemistry, chemistry, biology, and nutrition, along with engineers like myself whose specialty is detection instrumentation and biosensors.” Cunningham said; the center also involves clinicians from Mayo Clinic, Carle, OSF Hospital, Stanford, and Huntsman Cancer Institute “who could give us guidance about what kind of information would better guide their treatment decisions.  Our clinical partners also inform us about the shortcomings of currently available technologies—and help us target our work to where it can have the greatest clinical impact.”

The center was initiated in January 2020, and is planning a symposium to showcase its research goals later this spring.

https://www.igb.illinois.edu/article/new-center-genomic-diagnostics-will-reimagine-disease-detection-technologies