Researchers have developed a groundbreaking device that can diagnose glioblastoma, an aggressive brain cancer, in less than an hour. Using a biochip inserted into a small blood sample, the device detects epidermal growth factor receptors (EGFRs) in extracellular vesicles, using electrokinetic technology. This method offers high sensitivity and accuracy, reducing interference and improving early detection.
Glioblastoma is the most common and aggressive type of malignant brain tumor that affects adults. Originating in glial cells, which support and nourish neurons, glioblastoma grows rapidly and invades nearby brain tissue, making it especially difficult to treat.
The global incidence is approximately 3 to 4 cases per 100,000 people per year, and it is more common in men and people over 50.
Symptoms include persistent headaches, seizures, nausea, memory loss, vision changes, and cognitive or motor difficulties, depending on the location of the tumor in the brain.
Diagnosis is usually made by imaging tests, such as magnetic resonance imaging (MRI) or computed tomography (CT), and biopsies for histological analysis.
Treatments include surgery to remove as much of the tumor as possible, followed by radiation therapy and chemotherapy with temozolomide. Despite aggressive treatment, the prognosis is generally poor, with an average survival of 12 to 15 months after diagnosis. Although new advances, such as immunotherapy and targeted therapies, are being explored to improve outcomes, early diagnosis is the basis for a better prognosis.
Liquid biopsy is an innovative technique that offers a non-invasive way to diagnose cancer by detecting biomarkers present in blood or other body fluids. Instead of performing a traditional biopsy, which involves removing tissue directly from the tumor, liquid biopsy looks for small particles such as extracellular vesicles (EVs), exosomes, or even fragments of DNA and RNA that the tumor releases into the body.
EVs are tiny particles released by cells, including cancer cells, and carry within them materials such as proteins and nucleic acids (RNA and DNA), which can provide valuable information about the type and stage of cancer.
These vesicles are particularly interesting because they protect sensitive molecules, such as RNA, that would otherwise be rapidly degraded in the body. Because they are excreted in large quantities and are highly stable, EVs are seen as an excellent target for cancer diagnostics.
Researchers have identified that in tumors such as glioblastoma, cells release EVs with a specific molecular cargo, such as the Epidermal Growth Factor Receptor (EGFR), which can be detected through specialized tests.
A recent study published by researchers at the University of Notre Dame in the journal Nature focused on one such aspect, the specific epitope (a part of the protein) on the Epidermal Growth Factor Receptor (EGFR), which becomes more accessible in cancer cells due to molecular changes.
This epitope is present on both tumor cells and the extracellular vesicles they release, allowing cancer detection through a simple blood test. They analyzed 20 samples from patients with glioblastoma and 10 people without the disease.
The key to the diagnosis is a biochip that uses electrokinetic technology to detect biomarkers, or active epidermal growth factor receptors (EGFRs), which are overexpressed in certain types of cancer, such as glioblastoma, and found in extracellular vesicles.
The challenge for the researchers was twofold: to develop a process that could distinguish between active and inactive EGFRs and to create a diagnostic technology that was sensitive but selective in detecting active EGFRs in extracellular vesicles from blood samples.
Extracellular vesicles, or exosomes, are single nanoparticles secreted by cells. They are large, 10 to 50 times larger than a molecule, and have a weak charge. This technology was specifically designed for these nanoparticles, using their characteristics to our advantage.
To do this, the researchers created a biochip that uses an inexpensive electrokinetic sensor about the size of a ballpoint pen. Due to the size of the extracellular vesicles, antibodies on the sensor can form multiple bonds with the same extracellular vesicle. This method significantly increases the sensitivity and selectivity of the diagnosis.
The synthetic silica nanoparticles then “report” the presence of active EGFRs in the captured extracellular vesicles, while also carrying a high negative charge. When extracellular vesicles with active EGFRs are present, a voltage change can be seen, indicating the presence of glioblastoma in the patient.
This charge-sensing strategy minimizes interference common in current sensor technologies that use electrochemical reactions or fluorescence.
The detection method utilizes the highly negative zeta potential of silica reporters to produce a signal. a) General schematic and workflow of the platform. First, samples containing EVs are incubated, washed, incubated with silica reporters, and washed again. The platform consists of an anion exchange membrane that allows only counterions to pass through and exhibits three distinct regimes in the current voltage response. b) Current-voltage response of an anion exchange membrane: EVs produce no shift in the superlimitation region, while silica reporters produce a shift after forming a sandwich due to their highly negative charge. c) Automation algorithm of the platform with an automation interface, prototype, and biochip showing the AEM housing. d) Voltage shift for anti-CD63 capture and anti-CD63 reporter (both monoclonal and from the same clone) that target a specific epitope of CD63 binding only to species containing at least two unique copies of CD63 at different concentrations of sEVs measured in triplicates. e) Highly negative zeta potential of silica particles compared to EVs from two cell cultures and pooled healthy plasma (all done in triplicates except silica, where there are nine replicates).
For the test, blood is directly sampled without any pretreatment to isolate extracellular vesicles. This is because this sensor is not affected by other particles or molecules. It features low noise and makes it more sensitive for disease detection than other technologies.
In total, the device includes three parts: an automation interface, a prototype of a portable machine that administers materials to run the test, and the biochip. Each test requires a new biochip, but the automation interface and prototype are reusable.
It takes less than an hour to run a test, requiring only 100 microliters of blood. Each biochip costs less than $2 in materials to manufacture.
While this diagnostic device was developed for glioblastoma, the researchers say it could be adapted to other types of biological nanoparticles. This opens up the possibility for the technology to detect a range of different biomarkers for other diseases.
In addition, the current diagnostic platform could be expanded to test large libraries of untreated plasma from a large cohort of cancer patients to establish specific profiles for different types of cancer at different stages. Offering new hope for earlier and more accurate diagnoses.
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An anion exchange membrane sensor detects EGFR and its activity state in plasma CD63 extracellular vesicles from patients with glioblastoma.
Maniya NH, Kumar S, Franklin JL. et al.
Commun Biol 7, 677 (2024). https://doi.org/10.1038/s42003-024-06385-1
Abstract:
We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR, and its activity state, that provides a sensitive, selective, fluorophore-free, and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/μL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analyzed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and “active” EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.
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