Detecting Multiple microRNA Biomarkers of Alzheimer’s Disease

Alzheimer’s disease—an age-related neurodegenerative disease—is the leading cause of dementia. Alzheimer’s disease and related dementias are estimated to affect over 49 million people worldwide. It is characterized by several neuropathological changes, including neuroinflammation, neurodegeneration, and the build-up of extracellular amyloid beta plaques and intracellular tau tangles.

Alzheimer’s disease begins 20 years or more before symptoms begin, giving a significant window for early diagnosis and intervention to delay disease progression.

The Alzheimer’s Association has highlighted the importance of biomarkers in the approval of new treatments, for identifying people who may qualify for clinical trials, and in enabling earlier disease detection.

Hansana Gunathilaka is a graduate research assistant at the University of Connecticut who is currently working to develop a platform to detect Alzheimer’s disease biomarkers, under the guidance of Dr. James F. Rusling and Dr. Jessica L. Rouge.

At Pittcon 2026, Gunathilaka will discuss two CRISPR-Cas13a biosensing platforms that have been designed to detect multiple microRNA (miRNA) biomarkers of Alzheimer’s disease.

Finding biomarkers for Alzheimer's disease

Current approaches to identifying biomarkers for Alzheimer’s disease are based on brain imaging or analyzing cerebrospinal fluid (CSF) (Figure 1.).

Figure 1: Approaches to identifying biomarkers for Alzheimer's disease. Credit: AI-generated image created using Microsoft Copilot (2026).

MRI-based neuroimaging techniques, such as structural or functional MRI, reveal structural changes in the brain or alterations in blood flow. However, these changes may not be as evident in the earliest stages of disease progression, and these approaches are further limited by their accessibility to patients and the time spent in the scanner.

PET imaging allows the visualization of disease-associated molecules using ligands, but it is costly and presents risks associated with radiation exposure. Similar to MRI-based techniques, it is also hampered by availability and accessibility.

CSF analysis provides insights into protein production and clearance, particularly tau, phosphorylated tau, and amyloid beta. These biomarkers may be detected earlier than brain-based markers of Alzheimer’s disease. However, the lumbar puncture procedure to access CSF is often perceived as an invasive procedure.

Blood-based biomarkers are therefore more promising for routine use, as they are less invasive, lower in cost, and less time-consuming.

What led you to focus on detecting miRNA biomarkers of Alzheimer’s disease?

miRNAs are short, non-coding RNAs that influence gene regulation. They have been implicated in Alzheimer’s disease progression through their impact on amyloid-beta accumulation, tau aggregation, and neuroinflammation.

“We focused on circulating miRNAs because they are stable in blood, reflect key neurodegenerative processes, and show stage-dependent changes linked to Alzheimer’s pathology,” said Gunathilaka. “Our long-term goal is to translate this promising biomarker assay into low-cost, sensitive point-of-care diagnostic platforms usable in clinics and physicians’ offices.”

Using the new platforms, Gunathilaka and her colleagues detected the Alzheimer’s disease biomarkers miR-30e-5p, miR-34c-5p, and miR-200c-3p at femtogram per milliliter detection limits.

Key takeaways about Alzheimer’s disease diagnosis:

• Alzheimer’s disease has a long preclinical phase.
• Current diagnostic tools are often costly, invasive, or not sensitive enough to detect early molecular changes.
• miRNAs form promising blood-based biomarkers.

CRISPR/Cas biosensing platforms to detect miRNA

Can you explain in a little more detail how the biosensing platforms work and what drew you to use CRISPR/Cas13a in the platform?

Cas13a–CRISPR RNA complexes recognize a specific miRNA sequence, causing Cas13a to activate and convert to an enzyme. When Cas13a activates, it cleaves a reporter molecule that produces an optical signal, either fluorescence or electrochemiluminescence, which can be measured by a camera.

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“We were drawn to Cas13a because it offers exceptional specificity and signal amplification through enzyme turnover rather than nucleic acid replication,” Gunathilaka explained. “By integrating CRISPR with 3D-printed chips or well plates and electrochemical or fluorescence readouts, we created platforms that combine precision with scalable, user-friendly formats suitable for diagnostics.”

Key steps in biosensing platforms:

• CRISPR/Cas biosensors are activated by a specific target, which cleaves a reporter to produce a signal
• Cas13a offers high specificity and signal amplification for miRNA targets

Detecting multiple biomarkers

Why is enabling multiplex biomarker detection for Alzheimer’s disease so important, and what are the challenges facing the field?

“A single biomarker doesn’t capture the complexity of disease onset, progression, and patient-to-patient variability,” said Gunathilaka. She noted that relying on single-analyte tests can limit diagnostic accuracy and can make it difficult to track disease progression and therapeutic response over time. By contrast, multiplex biomarker detection allows more accurate tracking with enhanced precision.

“Key challenges include achieving ultrasensitive detection of low-abundance miRNAs without amplification, avoiding cross-reactivity for translating lab assays into easy-to-use, reproducible, clinically compatible platforms at low cost to the patient,” she outlined. “Our work addresses these challenges by combining multiplexing, CRISPR specificity, and signal amplification in essentially add-and-read assays.”

The benefits and challenges of multiplexing biomarker detection:

• Analyzing multiple biomarkers improves diagnostic accuracy and enables disease progression and therapeutic response tracking.
• Multiplexing faces several challenges, including low-abundance targets and avoiding cross-reactivity.

Using CRISPR biosensors beyond detecting miRNA

Can you tell us a little more about the next steps for your research? How will you approach developing CRISPR strategies to detect phosphorylated tau proteins?

“We want to incorporate two p-tau proteins, known Alzheimer’s biomarkers, into our CRISPR-based strategy,” said Gunathilaka.

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Levels of phosphorylated isoforms of the tau protein, including p-tau181, p-tau217, and p-tau231, have been shown to mirror pathological features of Alzheimer’s found in the brain.

However, with more than 40 distinct phosphorylation sites on tau, Gunathilaka and her colleagues must find a way to selectively isolate the isoforms of interest from blood or plasma samples.

“We are pursuing antibody–antigen interactions to couple their detection into our existing miRNA sensors to enable protein and miRNA detection simultaneously,” she explained.

What this means for the future of CRISPR-based strategies:

• Combining p-tau and miRNA detection would create a powerful diagnostic panel.
• Isolating disease-specific isoforms of p-tau poses challenges alongside detecting protein and miRNA simultaneously.

The research was conducted under the leadership of Dr. James F. Rusling and Dr. Jessica L. Rouge, with Dr. Rusling also reviewing Gunathilaka’s initial interview responses.

Hansana Gunathilaka would like to take this opportunity to acknowledge Dr. Keshani Hiniduma and Dr. P.I. Thilini De Silva, whose work laid the foundation for her talk at Pittcon 2026, and Rachelle Canete for their mentorship and guidance.