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Gold 'Nanodimple' Array With Plasmonic Sensor Lowers PCR Detection Thresholds, Researchers Say


NEW YORK – A research team in South Korea has developed a novel method to detect molecules for diagnostics applications. Using structures called nanodimples with internalized gold nanoparticles, the team was able to enhance signals and increase detection of target molecules.

Typically, PCR products are detected by methods like melting temperature, colorimetric dyes, or fluorescent signals. An alternative to these standard approaches is Raman spectroscopy, which requires no staining or labeling of the target molecules but instead detects changes in the way their vibrations scatter light.

Indeed, the method does not necessarily even require amplification. But, although it can be quite rapid and require little processing, it is hindered by low sensitivity.

To improve this, platforms using plasmon and surface-enhanced Raman spectroscopy have been in development for decades, according to a recent review of their use for virus detection. For example, surface-enhanced Raman scattering, or SERS, enhances signals through interaction with the electromagnetic field of a metal anchored to a surface.

The techniques offer enticing opportunities to improve detection thresholds in diagnostics, but to date they have also been challenging to manufacture in large scale.

In a proof-of-concept study published in Biosensors and Bioelectronics, Jaebum Choo and his colleagues at Chung-Ang University paired SERS with PCR and their nanodimple method to detect SARS-CoV-2 with higher sensitivity than other approaches.

The team fabricated arrays of nanodimples that had internalized gold nanoparticles using DNA hybridization. They then used these devices to perform surface-enhanced Raman scattering detection of PCR products and used bridge DNA probes that break down in the presence of target genes, such that the concentration decreases in the presence of the target.

Overall, they found that 25 RT-PCR thermal cycles were required to reach a detectable threshold for E gene and RdRp gene targets of the SARS-CoV-2 virus when the concentration started at 1 x 10^5 copies per microliter.

The number of cycles needed could be reduced to 15 by combining magnetic beads and SERS-PCR, but the targets could be detected at eight PCR cycles with their gold-internalized nanodimple method.

In an email, Choo said that by lowering the threshold for detection, the device can thereby decrease the time to result for a diagnostic test. For example, "It usually takes about 2 hours to run 40 cycles, so it takes about 24 minutes to run eight cycles," he said.

To date, in vitro diagnostic equipment using plasmonic sensing platforms has not been commercialized, Choo said. But his team is currently developing a portable Raman system combined with a lateral flow assay strip for which they plan to seek Korean Food and Drug Administration approval following clinical trials in the next three months. This diagnostic will be a nanoplasmonic sensing-based immunoassay system, he said, noting the team is also considering subsequent US FDA application as well. 

"We believe that the nanoplasmonics-based system can overcome the sensitivity limitation of fluorescence or luminescence detection techniques currently used in in vitro diagnostics," Choo said.

Giuseppe Spoto, a researcher at the University of Catania in Italy, is also developing plasmonic detection-based diagnostics. As previously reported, his team created a biosensor-based system to detect colorectal cancer using surface plasmon resonance imaging.

Specifically, Spoto and colleagues optimized a nanoparticle-enhanced surface plasmon resonance assay to skip PCR amplification entirely, he said in an email. The strategy eliminates biases and artefacts introduced by PCR amplification and simplifies the assay so that body fluids can be analyzed without pre-analytical processing.

However, the periodic array of plasmonic nanostructures used by Choo's team is "undoubtedly an interesting technology," Spoto said. He noted that a somewhat similar approach using silicon dioxide surfaces was first described in 2008, but that it has only recently been adapted for plasmonic sensing.

Spoto highlighted that large-scale production of arrayed plasmonic hot spots had been a challenge to the field. "We need technologies to produce such arrays with simple procedures and competitive costs," he said, adding that the method developed by Choo and his colleagues "may contribute to offering a suitable solution and developing competitive diagnostic approaches."

For the immunoassay project, Choo said that it should pose no problem for mass production because its manufacturing process is similar to that of a standard rapid immunoassay kit.

But, in the case of molecular diagnostics using PCR, the nanodimple approach is "a conceptually new device platform" to replace the existing fluorescence detection methods, he said.

To Spoto, the next step toward adoption of plasmonic diagnostics will be more application to real samples and a transition to commercialization.

In the future, "I believe that plasmonic-based diagnostics may significantly contribute to molecular in vitro diagnostics," he said.