Cancer Center at Illinois (CCIL) Program Leader Brian Cunningham has developed two new biomarker detection instruments with the potential to transform point-of-care diagnostics. In collaboration with researchers and clinicians at Stanford, Mayo Clinic, and Huntsman Cancer Institute, the Cunningham lab’s new instruments can vividly and rapidly detect a variety of cancer biomarkers using a novel gold nanoparticle tagging method.
“Nearly a decade ago, we invented a new biosensing instrument that incorporated our novel technology. We called it Photonic Resonator Absorption Microscopy (PRAM),” said Cunningham. “The original PRAM version we built was quite large, about four feet by eight feet, and cost approximately $200,000. It included an expensive microscope, spectrometer, cell incubator, and many top-of-the-line components. That first instrument did a great job of demonstrating the principle of PRAM, but the cost and size were prohibitive for clinical application. So, we’ve been working diligently to improve this technology for more practical applications in the lab and clinic.”
Cancer Center at Illinois (CCIL) Program Leader Brian Cunningham (left) and graduate student Weinan Liu present two new biosensing instruments for enhanced biomarker detection. The Cunningham lab’s new PRAM Mini makes dramatic improvements in cost, size, and user-friendliness compared to the original PRAM prototype developed a decade ago.
Cunningham lab’s AuNP-tagging PRAM technology is fundamentally different than any other biomarker detection method available.
As opposed to a typical glass slide, the PRAM technique employs a photonic crystal as a surface for microscopy. The photonic crystal provides very high contrast for detecting and counting individual gold nanoparticles (AuNPs). The AuNPs are tags deposited on molecules for biosensing, enabling researchers to observe and count individual molecules in a sample. (See Figure 1 for explanation of nanoparticles.)
“In some cases, the molecule we may want to detect could be a protein, a nucleic acid molecule called micro-RNA, or a circulating tumor DNA. In other cases, we can detect antibodies, too,” said Cunningham. The potential for beneficial clinical application is far-reaching. Presently, the clinical researcher pioneering PRAM’s application is Cunningham’s collaborator Dr. Manish Koli at Huntsman Cancer Institute. Dr. Koli is driving the large genomic studies on prostate cancer patients, identifying specific biomarkers to differentiate between aggressive and non-aggressive cancer, and monitoring changes in biomarkers over the course of treatment.
Version 1: The Automated Portable PRAM (ap-PRAM)
Cunningham’s lab has been working diligently to reduce PRAM’s size, cost, and user-burden, and they are pleased to report two new PRAM instruments that accomplish this vision.
The first is an automated, portable version of PRAM named ap-PRAM. This novel instrument is reported in a recently published paper in the journal Biosensors and Bioelectronics.
PRAM is no longer bigger than a ping-pong table. The new ap-PRAM is roughly the size of a toaster oven with components costing about $12,000. Cunningham estimates it could be manufactured at scale with a cost of only $5,000. The ap-PRAM instruments’ advances include rapid biomarker detection, autofocusing, multiplexing, millimeter-scale tiled field of view, dynamic analysis of binding interactions, improved image processing algorithm, increased noise suppression, and a simplified representation of AuNPs’ binding and binary appearance in the enhanced digital resolution.
The ap-PRAM user doesn’t have to fiddle with knobs or manual adjustments, thanks to the microscope’s autofocusing which provides sharp display of AuNPs. The ap-PRAM can move the biosensor, too, by tiling together many fields of view and obtaining a larger surface area where all the AuNPs can be counted. It is also able to perform multiplexing—accommodating and measuring many biosensors in a single cartridge in rapid succession. Notably, ap-PRAM can also train its attention to one small point, measuring AuNPs accumulating there as a function of time. By studying the accumulation rate, this allows the user to ascertain the minimum time needed for accurate detection, rather than waiting for a full measurement of molecules.
“If the required information can be gathered in just five minutes, this time saved will greatly improve the point-of-care experience for both patient and user,” added Cunningham.
Version 2: The PRAM Mini
Because the cost and size of ap-PRAM would still be prohibitive for certain users, Cunningham’s lab wanted to push the boundaries even further. With significant efforts by graduate student researchers Kodchakorn Khemtonglang and Weinan Liu, the research team developed PRAM’s next iteration, the PRAM Mini. The new technology is reported in a recently published paper in the journal Biomedical Optics Express.
The PRAM Mini increases user-friendliness and dramatically reduces the form factor and costs of the original PRAM. Cunningham reports the PRAM Mini is the size of a textbook with an estimated manufacturing cost of only $500.
The PRAM Mini prototype is built on laser cut, quarter inch thick acrylic sheets, with holes in which are mounted a 3D printed structure to hold the optical components. The PRAM Mini eliminates the additional automated features of the ap-PRAM but is still capable of same limits of biomarker detection in other PRAM’s, using the team’s sophisticated AuNP tagging method. The PRAM Mini employs the same optical components as ap-PRAM but uses a Raspberry Pi microcomputer controller to communicates via Bluetooth with a linked mobile device.
“The PRAM Mini is very user-friendly, could easily go into a doctor’s office, and accomplishes detection within just five minutes. We believe it will revolutionize the future of point-of-care diagnostics for a range of different diseases,” Liu said.
While the original PRAM instrument provided high-contrast images, the more portable, user-friendly versions do come a sacrifice in image quality. However, with the incorporation of sophisticated machine learning algorithms, the research team is refining the technology so that affordability and portability don’t translate to a reduction in detection resolution.
A screen capture of PRAM’s digital biosensing. Anywhere there’s a AuNP, it absorbs red light and PRAM displays a little dark spot in the image. Instead of seeing many millions of AuNPs in aggregate, PRAM can count the individual molecules for unprecedented, accurate biomarker measurement.
The Future of PRAM
In addition to continue to fine-tune the computational algorithms for maximal image resolution, the research team is considering additional improvements to the newest iterations of PRAM. “Because we want to keep the size and cost as minimal as possible without sacrificing ultimate biomarker detection efficacy, our next endeavor will be to work on the inclusion of multiple biomarker detections within a single image field of view—that is, maximizing information density given our existing constraints,” commented Cunningham.
In the future, when sitting in the doctor’s office, your care provider might use the non-invasive PRAM technology to perform accurate, cost-reducing cancer diagnostics within just five minutes. Your doctor might determine whether prostate cancer, for example, is an aggressive or less aggressive form. Or, your physician may use PRAM for treatment monitoring, assessing the efficacy of your treatment levels and deciphering any possible mutation recurrences that slipped by previous treatments.
Cunningham’s Product Development Ventures
To help distribute this new biosensing instrumentation at a larger scale, Cunningham formed a new company, Atzeyo Biosensors, with the aim “to change the lives of cancer patients” by developing innovative point-of-care diagnostic and monitoring platforms. Their innovative point-of-care technology will enable physicians to receive critical diagnostic results in real-time, in the office. Founded in 2023, Atzeyo recently completed an initial $300,000 round of seed funding to develop PRAM Mini into a commercial product. The company has organized a team of experts in diagnostics and detection instrument manufacturing, finance, and regulatory measures. Atzeyo will soon hire a product development company to take the initial PRAM Mini concept and design for manufacturability and user interface.
Summary
Cunningham’s innovative ap-PRAM and PRAM Mini instruments are fundamentally different than any existing biosensing technology for biomarker detection. PRAM employs a simple, low-input biochemistry method to label each molecule in the sample with a gold nanoparticle, enabling quick, accurate results. This technology holds great promise for cancer diagnostics and monitoring, as it can detect various cancer biomarkers, including proteins, microRNAs, and circulating tumor DNA. Finally, PRAM technology has the potential to transform point-of-care settings because of its low-cost instrumentation, simply assay method, and the high sensitivity obtained through the unique digital counting method.
“There’s nothing else that does what we are trying, so we think we have something special,” concluded Cunningham.
Editor’s notes:
The ap-PRAM is reported in the paper “Dynamic and large field of view photonic resonator absorption microscopy for ultrasensitive digital resolution detection of nucleic acid and protein biomarkers” and is available online.
doi.org/10.1016/j.bios.2024.116643
The PRAM Mini is reported in the paper “Portable, smartphone-linked, and miniaturized photonic resonator absorption microscope (PRAM Mini) for point-of-care diagnostics” ad is available online.
Brian Cunningham is CCIL Program Leader for the Cancer Technology and Data Science (CTD) program, the Intel Alumni Endowed Chair of the Department of Electrical and Computer Engineering, a professor of bioengineering, and the Holonyak Micro and Nanotechnology Lab. Cunningham is also the Director of the Center for Genomic Diagnostics theme at the Carl R. Woese Institute for Genomic Biology.
To contact Brian Cunningham, email bcunning@illinois.edu
This story was written by Jonathan King, CCIL Communications Specialist.
