A team of researchers at the University of California Los Angeles (UCLA) has developed a method to speed and simplify detection of proteins in blood and plasma, opening up the potential for early infectious disease detection or cancer during a doctor's office visit. The new test, which takes about 10 minutes as opposed to two to four hours for current tests, is promising for use with portable optical readers, such as smartphones.
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The new approach overcomes several key challenges in detecting proteins that are biomarkers of disease. First, these proteins are often at low abundance in body fluids and accurately identifying them requires amplification processes. The current approach uses enzymes to amplify the signal from proteins. However, enzymes can break down if they are not stored at proper temperatures. Also, to avoid a false positive, excess enzymes need to be washed away. This increases the complexity and cost of the test.
The researchers included lead author Donghyuk Kim, a UCLA postdoctoral researcher in bioengineering and Dino Di Carlo, professor of bioengineering at the Henry Samueli School of Engineering and Applied Sciences. They collaborated with Aydogan Ozcan, Chancellor's Professor of Electrical Engineering and Bioengineering and Omai Garner, assistant professor of pathology and medicine at the David Geffen School of Medicine at UCLA.
The researchers devised an approach to amplify a protein signal without any enzymes, thus eliminating the need for a complex system to wash away excess enzymes, and that would work only in the presence of the target protein. This new approach made use of a molecular chain reaction that was strongly triggered only in the presence of a target protein.
The molecular chain reaction is driven by a cycle of DNA binding events. The process begins with a DNA key divided into two parts. If the target protein is present, the two parts bind together to form a DNA complex. The formation of the DNA complex generates DNA signaling molecules, which in turn generates the same DNA complex, leading to more signaling molecules and thus propagating repeated cycles.
Unlike previous approaches to achieve an amplified readout of proteins, such as the proximity ligation assay, this approach does not require multiple enzymes, longer polymerization-based enzymatic reactions, or temperature control to amplify signal, according to Di Carlo. The team's new assay, he says, operates at room temperature and achieves results in about 10 minutes.
|UCLA researchers were able to use a molecular chain reaction to detect the presence of proteins in blood and plasma in a way that is faster and simpler. (Image credit: Donghyuk Kim/UCLA)|
They demonstrated the approach with two target proteins—streptavidin, widely used as a test protein for new diagnostic assays, and influenza nucleoprotein, which is a protein associated with the influenza virus. In the long term, the team aims to combine the technique with portable readers that could be particularly beneficial in clinics in resource-poor areas.
"Because the technique requires fewer steps than other assays, it can have a significant impact on distributed diagnostics and public health reporting, especially in combination with cost-effective portable and networked reader technology that our lab is developing," Ozcan says. His team's handheld microplate reader is suitable for protein diagnostic assays based on a smartphone's optical and computational systems.
Garner, who is also the associate director of the clinical microbiology lab at UCLA Health, emphasized the broad application of the technique. "Although demonstrated initially in detecting protein associated with flu, we envision the approach can be generalized to a range of protein biomarkers associated with infectious diseases and cancer," Garner says, noting that it could be configured to detect Zika or Ebola viruses.
The researchers emphasized that additional work is required to adapt the assay to complex clinical samples that may have other interfering compounds, and further optimization of the reagents for the assay can enhance performance.
Full details of the work appear in the journal ACS Nano; for more information, please visit http://dx.doi.org/10.1021/acsnano.6b02060.