NEW YORK (GenomeWeb) – Researchers at the University of New South Wales have developed a magnetic nanoparticle-based technique to directly detect microRNA in unprocessed whole blood samples in about thirty minutes.
Researchers currently use quantitative reverse-transcription PCR (qRT-PCR) to profile miRNA due to its usually high specificity and sensitivity. Although qRT-PCR produces reliable results, the researchers noted that it does not detect miRNA directly in whole blood. Instead, researchers perform a lengthy process where they must isolate and purify RNA, and then synthesize cDNA to measure miRNA expression.
"Quantitative PCR normally takes up to 10 hours, while our method only takes about half an hour to produce results" John Gooding, a chemistry professor at University of NSW, Sidney explained.
In a study published yesterday in Nature Nanotechnology, Gooding and his team used gold-coated magnetic nanoparticles (Au@MNPs) modified with redox-labeled DNA probes to detect expression levels of miR-21 — a mRNA that can act as a cancer biomarker due to its ability to inhibit tumor suppressor genes — in whole blood samples.
First, the researchers modified the surface of the Au@MNPs with a probe DNA sequence complementary to miR-21 and marked it with a blue redox label. The team then added excess "DNA-Au@NMPs" to the analyte solution. After 30 minutes, the team magnetically separated the Au@MNPs from the solution and washed away unhybridized sequences.
Gooding and his team then collected the Au@MNPs on the surface of a gold microelectrode using a magnet. The team removed the magnet and applied 10 cycles of square-wave voltammetry between 200 and 500 megaVolts, with a pulse amplitude of 20 megavolts and frequency of 2 hertz. The team then used stable peak currents obtained before and after hybridization to measure the amount of analyte.
To examine the electrochemical tool's limit of detection and range of its sensor, the team prepared different concentrations of miR-21 in either phosphate buffered saline (PBS), undiluted human serum, or 50 percent whole human blood. Gooding and his group measured a change in the current after 30 minutes of exposure to the miR-21 solutions within the concentration range of 10 attomolar to 1 nanomolar.
Despite the tool's impressive limit of detection, Gooding explained that his team did not initially plan to find a target in the attomolar range. His team originally wanted to demonstrate that the tool could detect miRNA in a concentration range of 10 centimolar to picomolar, yet their expression levels above and below that range indicated a whole variety of conditions.
"When performing the calibration, we were going to be ecstatic if we got to the standard range of detection," Gooding said. "The fact that we got to attomolar — [which] we reported not because that was the goal, but because that was what the technology could actually achieve — was quite surprising."
In addition to the assay's speed and high sensitivity, Gooding noted that "what makes [th
While Gooding and his team do not fully understand how the rearrangement occurs on the magnet's surface, they theorize that it is caused by some combination of duplex creation and electron reconfiguration.
In order to validate the electrochemical tool, the researchers used it to analyze miR-21 levels in a pool of other RNA sequences from the total RNA extracted from human lung cancer cells (A459) and exosomes. After treating the samples with with an miR-21 inhibitor, the team still detected levels of miR-21 in exosomes eight times lower than the factor of 0.4 in their parental cells.
"Given that the sensor was able to detect such marked variations in the levels of miR-21 expression suggests that it has potential as an accurate and selective method for analyzing variations in the expression levels of miRNA in the presence of a pool of other RNA sequences," the authors said.
To further examine the sensor's ability to directly detect miR-21 in clinical samples, the team spiked different amounts of RNA from A549 cells into PBS or 50 percent whole human blood. They found out that the electrochemical sensor could selectively identify miR-21 found directly in human blood without prior extraction.
Finally, Gooding and his team demonstrated the tool's clinical utility by measuring miR-21 expression in whole unprocessed blood in a mouse model of human lung cancer.
Using the DNA-Au@MNP-based electrochemical sensor, the team found that the concentration of miR-21 molecules in rapidly growing cancers was higher than those in the "no-control" group. The group also noted that the electrochemical assay had less variability than qRT-PCR with "no requirement for laborious sample processing and RNA extraction."
Gooding explained that his team decided to use gold over other metals because of its stability, conductivity, and ability to "be easily modified." Highlighting that the gold particles themselves are not the expensive element of the tool, Gooding noted that the time to develop the particles was instead relatively expensive.
"Gold is a very stable metal, as it doesn't oxidize in ambient conditions, and instead maintains many of its properties," Gooding said. "Gold can bind very easy to the vials, and because the nucleic acids can be diluted, you can modify [the metal] very easily."
Gooding acknowledged that his team has encountered technical issues while working on the tool, including reliable nanoparticle creation and examining potential applications for the sensor. According to Gooding, developing the electrochemical tool to efficiently synthesize and bind to miRNAs has always been tricky, especially on such a small scale, "but it's something we're working on now to improve" for future applications.
In addition, Gooding believes that figuring out the tool's potential utility in different areas will be a challenging prospect. He noted that the group wants to "expand the range of fields, from nanoparticle synthesis and modifications to putting on nucleic acids …. [as well as] performing cell-based studies, and even using animal samples in [the assay]."
In the future, Gooding's team will begin studies to further understand the mechanism behind the electron binding process. He explained that while his team had previously used a similar approach to detect proteins, the researchers could not "identify the second part of the mechanism."
In addition, the authors noted that "this sensing concept could be tested for obtaining information about the expression profiles of other circulating markers such as circulating DNA or proteins in whole blood, in parallel with circulating miRNAs."
While the technology is currently early in development, Gooding noted that if the tool remains promising, his team would pursue a commercial path in the future.
"This [tool] would be especially useful for low levels of certain proteins and molecules, as we could [potentially] not only diagnose a patient's condition, but [also] look at treatment efficacy or the potential relapse of a condition [like] cancer," Gooding said.
While he is unaware of companies that are using similar technology to look at microRNAs, Gooding noted that University of Toronto researchers have developed an electrochemical clamp assay to detect circulating nucleic acids.