A newly developed technique could make it easier to measure very small concentrations of molecules, which may have implications in early diagnostic testing for Alzheimer’s and other diseases.
The technique was described in Research, in the study “Single-Atom Nanozymes Linked Immunosorbent Assay for Sensitive Detection of Aβ 1-40: A Biomarker of Alzheimer’s Disease.”
One of the most common techniques used in biomedical laboratories is the enzyme-linked immunosorbent assay (ELISA). The test allows researchers to measure the amount of a given target molecule — for instance, a disease marker — using antibodies that specifically recognize and bind to that target molecule. The use of antibodies, which are immunological proteins, is where the “immunosorbent” part of the assay’s name comes from.
Antibodies cannot be seen binding to their targets, so the antibody in an ELISA is attached to an enzyme that, when exposed to certain substances, creates a color change. Thus, researchers can indirectly measure how much of the target is present by the extent of color change induced by the enzyme.
Usually, the color-changing antibody used in ELISAs is horseradish peroxidase (HRP), which is found in the horseradish plant.
While HRP-based ELISAs have been effectively used in research, the HRP enzyme is not perfectly efficient. As such, these ELISAs can’t reliably detect very small quantities of their targets.
This limitation poses a problem in diagnostics. For example, in Alzheimer’s, the protein amyloid beta 1-40 (Aβ 1-40) in the blood is thought to be a disease marker. This protein may be present in the blood at very early stages of the disease but at such low concentrations that ELISAs cannot reliably detect them.
“Detecting Aβ 1-40 at low concentration is of great importance and can be used to estimate the risk or show the presence of [Alzheimer’s] at early stage,” the researchers wrote.
The team designed an artificial enzyme called a single-atom nanozyme (SAN), composed of single iron ions embedded in nitrogen-coated carbon nanotubes. The SAN functions similarly to HRP but, since the SAN was designed by people, whereas HRP is the product of evolution, the SAN is more efficient. The SAN is also more resistant to changes in temperature and acidity than the natural enzyme.
“The nanozyme based on a single-atom catalyst that we created has a similar structure as a natural enzyme with remarkable enzyme-like activity,” Annie D. Du, PhD, study co-author and a professor at Washington State University, said in a press release.
The researchers then incorporated their SAN into an immunoassay functionally identical to an ELISA, which they called a SAN-LISA (SAN-linked immunosorbent assay).
The SAN-LISA was able to detect Aβ 1-40 levels as low as 0.88 picograms (pg)/mL. This was more than 10 times lower than the limit of detection for a commercial ELISA kit (9.98 pg/mL).
“This shows great potential for the early-stage diagnosis of Alzheimer’s Disease,” Du said.
Beyond Alzheimer’s disease, this technique could “pave a new way to design ELISA kits with improved sensitivity for detecting various target biomarkers,” the researchers concluded.
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