Glass nanopores pull DNA through a needle like spaghetti

DNA sequencing has become so common that few realize the difficulty of extracting a single DNA molecule from a biological sample.

Research led by UC Riverside makes it easy to detect and collect DNA from fluid samples such as blood using a tiny glass tube and electric current. The technique described in the diary, Nanoscalecan also improve cancer diagnosis in the future.

DNA, a double-stranded, electrically charged molecule that contains all the information an organism needs to create and organize the building blocks of life, is tightly folded in the cell nucleus. Extracting DNA from a single cell is time consuming and impractical for many medical and scientific purposes. Fortunately, as the cells naturally die, their membranes burst, releasing their contents, including DNA. This means that a blood sample, for example, contains many strands of free-floating DNA that, in theory, should be easier to identify and extract in quantities.

However, scavenger cells called macrophages, which clear up cell waste, destroy most cell-free DNAs, leaving them in low concentrations in the blood. Most approaches to capturing cell-free DNA require expensive techniques that first concentrate the molecules before using fluorescent dyes to better identify the DNA.

Corresponding author Kevin Freedman, an assistant professor of bioengineering at UC Riverside's Marlan and Rosemary Bourns College of Engineering, worked to improve the detection and detection of DNA at lower concentrations by placing an electrically charged DNA sample directly into a glass tube with a tiny opening called the a nanopore was directed. Nanopore detection has proven to be a fast, reliable, and inexpensive diagnostic tool for various medical and clinical applications.

"We know that when you apply voltage to a cell membrane, ions move through pores in the cell membrane," Freedman said. "DNA also moves with the electric field, and we can use it to move DNA."

The researchers inserted a positive electrode into a glass tube with an opening or pore 20 nanometers wide – slightly larger than a DNA molecule, but too small to hold cells. They applied an electrical potential to the nanopore, which was immersed in a vial containing a DNA sample and a negative electrode. The cell-free DNA moved into the pore and blocked it. The change in the electrical current as the DNA moved through the pore enabled the researchers to detect it.

An illustration shows (a) how a glass tube with a tiny opening and a positive electrode, when inserted into a liquid sample and stimulated with electricity, collects cell-free DNA floating in the sample; (b) a photograph of the borosilicate glass nanopore (Freedman et al. 2021

"It's like trying to pull spaghetti through a needle," Freedman said. "To go through the pore, it has to be almost perfectly linear."

The closer the researchers held the pore to the surface of the liquid, the more DNA it took in.

“Amazingly, we found that DNA accumulates at the interfaces between liquid and air. When there's a cooling layer, the DNA tries to get to the cooler place, ”Freedman said. “We hope that this will also apply to a blood sample, so that the same mechanism can be used to concentrate DNA near the surface. Not only is this beneficial, but this nanopore capture strategy also showed a higher signal-to-noise ratio near the surface. It really is a win-win situation. "

With some improvements, the authors believe their all-electric technique could help diagnose some types of cancer from a single blood sample. In addition to DNA, vesicles are released into the bloodstream as tumors grow. These mini-lipid-based droplets can be viewed as mini-cells, which are identical to the original cancer cells and can also be detected by nanopore sensing.

In view of all the unique features of this purely electrical technology, the nanopore sensor system can in future be used as a diagnostic test evaluation at the treatment site.

Source: UC Riverside

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