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Singapore's Star Array Prepping to Launch Microfludic Chip for Single-Cell NA Purification, qPCR


Star Array, a spinout of Singapore's Nanyang Technological University, has developed a microfluidic liquid-phase nucleic acid purification chip that can selectively isolate DNA or RNA from low numbers of cells in minute sample volumes, and subsequently PCR amplify the purified nucleic acids on chip.

The company believes its technology has applications similar to those enabled by digital PCR: single-cell gene expression, low copy number detection, environmental testing, and, eventually, point-of-care molecular diagnostics, Hai-Qing Gong, an associate professor at NTU and co-founder of Star Array, told PCR Insider recently.

In addition, Star Array is developing a suite of instruments, including a fluidic processor and qPCR machine, to complement the microfluidic chip and enable "all-in-one-well" genetic testing, and aims to make the instruments commercially available in the second half of this year, Gong said.

To that end, the company recently published a pair of proof-of-concept papers describing its platform and demonstrating various genetic analysis applications for it.

First, in December, Gong and colleagues published a paper in Analytical Chemistry demonstrating how its microfluidic chip could be used to selectively liquid-phase extract DNA or RNA from bacterial cells in the range of 5,000 down to a single cell from minute sample volumes, then perform on-chip quantitative PCR on the nucleic acids.

Then, in a research note published last month in Analytical and Bioanalytical Chemistry, the researchers used their biochip to quantify, via a real-time PCR assay, bacterial adhesion to single host cells, thus demonstrating a technique for analyzing host-pathogen interactions at the single-cell level without the use of fluorescence labels.

Of these papers, the former was the first general description and demonstration of Star Array's platform.

In an email to PCR Insider, Gong noted that his team developed the nucleic acid purification chip to address shortcomings in current methods of preparing nucleic acids for downstream analysis from tiny sample volumes.

"Many clinical and environmental testing applications require [the analysis of] DNA or RNA without interference of other [RNA or DNA]," Gong said. "For example, testing of RNA can be used [to determine] viability of bacteria. Also, for single-cell gene expression in which we don’t wish to get noise from [background] DNA."

Gong said that when it comes to purifying nucleic acids from minute sample volumes, existing commercial solutions such as solid-phase extraction-based kits often don't cut it, requiring sample volumes of between 50 µl and 200 µl.

"Our method can recover nucleic acid from 1 µl or even 100 nl volume sample," Gong said, noting that a given sample volume is as small as the volume of a single well on the microfluidic chip.

"This may open many new applications in clinical and environmental testing, including testing one-drop blood sample[s] from [piercing] one’s finger, which may make … POC genetic testing truly bedside [for] cancer diagnostics, forensics, et cetera."

In their Analytical Chemistry paper, Gong and colleagues used their chip to purify nucleic acids from Pseudomonas aeruginosa and Staphylococcus aureus, model gram-negative and gram-positive bacteria, respectively.

Bacterial cells were probabilistically isolated in the wells of their chip, much in the same way that droplet-based digital PCR relies on Poisson distribution to ensure that single copies of target nucleic acid are isolated in individual droplets for subsequent amplification.

"If one cell is isolated in each well, we can do absolute quantitation of the cells in a sample [like digital PCR]," Gong said. "If we isolate more than one cell in a well, we can do qPCR in each well. This makes our platform versatile."

The bacterial cells can be lysed to release their nucleic acid using a variety of methods — [it could] be enzyme and thermal lysis, [or] other physical lysis [methods] like ultrasound," Gong noted.

Using their chip, the researchers were able to recover nucleic acid from multiple or single bacterial cells at an average of 10-fold higher yield than commercial column-based solid-phase extraction methods.

Further, the group described the integration of the chip with Star Array's StarCycler fluorescence-based instrument for quantitative PCR, which contains multiple integrated functions including thermal cycling control, real-time fluorescence imaging, and data analysis.

Using this combined platform, Gong and colleagues were able to demonstrate high-throughput extraction of RNA followed by direct on-chip PCR analysis from single bacterial cells.

Meantime, in their Analytical and Bioanalytical Chemistry paper, the researchers used their platform to profile the association of P. aeruginosa to single host human lung epithelial cells, in the process revealing an adherence distribution that they claim had not been previously reported.

In both papers, the researchers described using a 900-microwell two-dimensional array chip with vacuum-driven microfluidics.

Gong said that both papers demonstrate a key aspect of the technology: its ability to purify nucleic acid and conduct PCR within the same chamber, thereby "eliminating any potential loss of nucleic acid between different sample prep steps (e.g. loss in elution step when using SPE) and transfer processes (e.g. from [the] sample prep station to PCR station)." This capability, he added, is important for single-cell analysis and other low copy number applications.

In general, Star Array in the near term is aiming to commercialize its platform for sample prep in nucleic acid analysis and single-cell genetic analysis. "The company is also developing this platform for digital PCR, digital cell PCR, and PCR arrays for parallel gene analysis," he said.

Star Array has obtained a license from NTU to several patents covering the technology, Gong added. The company is currently testing additional sample types on its system, and is seeking partners to explore different applications to the technology.