By Ben Butkus
Chinese scientists have developed a prototype device that integrates nucleic acid purification, isothermal amplification, and fluorescence detection on a single glass chip, and have demonstrated its ability to purify and detect DNA from complex biological matrices such as blood.
According to the researchers, the device performs DNA extraction as well as or better than a commercial kit from Qiagen, is less expensive than other integrated lab-on-a-chip devices, and can provide results in less than two hours, making it ideal for potential point-of-care molecular diagnostic applications.
The scientists, from Zhejiang University in Hangzhou, China, published details of the development and initial testing of their device in a paper this month in Analytical Chemistry.
"The purpose [of] our research is to develop a fully integrated genetic analysis system and promote the development of point-of-care systems," Ying Mu, corresponding author on the paper, said in a recent e-mail to PCR Insider.
As described in the paper, the prototype device consists of a 1-mm-thick, 63-by-63-mm glass plate with a chromium and photoresist coating, as well as microstructures fabricated using photolithographic and chemical etching techniques.
The integrated system contains two distinct functional domains, one for nucleic acid extraction and another for a loop-mediated isothermal amplification, or LAMP, reaction.
The extraction domain contains a sample inlet; a glass solid-phase extraction bed consisting of a serpentine channel with an array of 50 µm-diameter micropillars; and an extraction waste arm. The LAMP domain contains a LAMP reagent entrance channel, an amplification chamber, and a LAMP outlet.
The researchers wrote in their paper that they chose glass alone as the material for the nucleic acid purification unit because it was much simpler to fabricate and more conducive to PCR temperature cycling than many previous lab-on-chip technologies employing silicon beads or sol gel.
While other technologies have used microfabricated silica pillars to increase the surface area available for DNA capture, Mu and colleagues designed glass micropillars for the same purpose.
The use of LAMP instead of conventional PCR further simplified the system, primarily because it can amplify specific DNA isothermally using a water bath or heating plate as opposed to a more expensive thermocycler.
"Another important advantage of LAMP is its simplicity for detection of the amplification reaction," the researchers wrote, noting that a positive LAMP reaction yields a visible white precipitate, the detection of which can be enhanced by adding a fluorescent intercalating dye such as SYBR Green.
Finally, the prototype device contains drilled access holes at the ends of the channels, an identical glass cover plate bonded to the etched bottom plate, and stainless steel tubes inserted into the access holes, thus creating a closed system.
To test the ability of their device's glass solid-phase extraction component to extract DNA, the researchers compared its efficiency with that of a commonly used DNA extraction kit, the QIAamp DNA kit from Qiagen.
They used both technologies to extract DNA from whole blood samples. Based on visual inspection of gel electrophoresis bands following extraction, the researchers concluded that "DNA extraction using the gSPE bed is not worse than that using the commercial kit or even may be better."
Mu told PCR Insider that the group will likely be able to further improve extraction "by extending the channel length [to create a] larger surface area. However, [this] is not a good idea for an integrated, compact genetic analysis system. We are therefore making some other efforts to get better results."
Next, the researchers demonstrated that DNA extracted using the gSPE bed could be amplified using LAMP in a separate PCR tube; and finally, they used their integrated device to successfully extract, amplify, and detect lambda phage DNA in less than two hours.
All of this was performed without expensive or complicated instrumentation, the group noted. "We need [a] microscope to observe the fluidic direction during the operation of flow control," Mu said. "A hot plate was used for the LAMP reaction; and equipment with the [ability to perform] fluorescence imaging and fluorescence intensity readout was required to analyze the LAMP reaction."
The researchers noted that their prototype device is a long way from commercialization – for instance, "some details such as automatic operation and stability need to be optimized," Mu said. However, they believe that it has potential in myriad applications, not least among them point-of-care diagnostics.
"Our original intention was to develop a fully integrated genetic device to promote the development of point-of-care [testing], which includes the field of molecular diagnostics," he said.
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