Abstract
Cell‐free nucleic acids (cfNAs) are emerging diagnostic biomarkers for monitoring the treatment and recurrence of cancers. In particular, the biological role and clinical usefulness of cfNAs obtained from the plasma of patients with various cancers are popular and still intensely explored, yet most studies are limited by technical problems during cfNA isolation. A dimethyl dithiobispropionimidate (DTBP)‐based microchannel platform that enables spontaneous cfNA capture in 15 min with minimal cellular background and no requirements for use of bulky instruments is reported first. This platform identified KRAS and BRAF hot‐spot mutations following cfDNA isolation from the blood plasma and tissues obtained from 30 colorectal cancer patients. The correlation of mutations between the primary tissues and plasma from the patients was high using this platform with whole genome sequencing compared to the spin‐column method. This platform can also be combined with various detection approaches (biooptical sensor, Sanger sequencing, and polymerase chain reaction (PCR)) for rapid, simple, low‐cost, and sensitive circulating tumor DNA detection in blood plasma. The efficiency and versatility of this platform in isolating cfNAs from liquid biopsies has applications in cancer treatment and precision medicine.
Highlights
One major technical issue in cfDNA analyses is the efficiency of the extraction procedure in obtaining the DNA from plasma
The cell-free nucleic acids (cfNAs) isolation assay includes four steps: 1) chip surface modification, 2) sample mixing, 3) binding, and 4) washing and elution steps that can be performed in a single DTBP platform (Figure 2)
Use of the DTBP platform without a cell lysis buffer and instruments (Figure 1A) allows the isolation of cfNA from blood plasma within 15 min by overcoming the limitations of the columnbased method, such as the increased cellular background owing to cell lysis, the requirements of chaotropic reagents, large sample volume, and the use of instruments
Summary
One major technical issue in cfDNA analyses is the efficiency of the extraction procedure in obtaining the DNA from plasma. Recent cfDNA isolation methods that do not use centrifuges have been developed, they still need additional instruments, such as vacuum systems and DC power supplies for fluorescence labeling.[13–15] To overcome this issue, procedures of cfDNA sampling from blood plasma need to be standardized to obtain a sufficient amount of DNA and reduce the cellular background, which would subsequently improve the detection sensitivity of ctDNA mutation profiling. Compared with the column-based method, this DTBP platform rapidly isolates cfNA within 15 min and does not require bulky instruments (e.g., a centrifuge or a vacuum pump) We applied this DTBP platform to compare analyzed plasma with analyzed tumor tissue performed by KRAS and BRAF testing in 14 prospective colorectal cancer (CRC) patients (stages I–IV) and in 10 healthy controls. This new platform offers a rapid, simple, low-cost, and reproducible blood-based profiling test
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