Researchers have spent more than three decades developing and studying miniature biosensors capable of identifying single molecules. In five to ten years, when such devices could become a staple in doctors’ offices, they could detect molecular markers of cancer and other diseases and assess the effectiveness of drug therapy in combating these diseases.
To achieve this and to increase the accuracy and speed of these measurements, scientists must find ways to better understand how molecules interact with these sensors. Researchers at the National Institute of Standards and Technology (NIST) and Virginia Commonwealth University (VCU) have now developed a new approach. They reported on their findings in the current issue of Scientific advances.
The team built their biosensor by making an artificial version of the biological material that forms a cell membrane. Known as the lipid bilayer, it contains a tiny pore about 2 nanometers (billionths of a meter) in diameter, surrounded by fluid. The ions dissolved in the fluid pass through the nanopore, generating a small electric current. However, when a molecule of interest is embedded in the membrane, it partially blocks the flow of current. The duration and extent of this blockage serves as a fingerprint, identifying the size and properties of a specific molecule.
To perform accurate measurements for a large number of individual molecules, the molecules of interest must remain in the nanopore for an interval that is neither too long nor too short (the “Goldilocks” time), ranging from 100 millionths to 10 thousandths of a second. The problem is that most molecules only stay in the small volume of a nanopore during this time interval if the nanopore holds them in place. This means that the environment of the nanopores must provide some barrier – for example, adding an electrostatic force or changing the shape of the nanopore – that makes it harder for molecules to escape.
The minimum energy required to cross the barrier differs for each type of molecule and is essential for the biosensor to function efficiently and accurately. The calculation of this quantity consists in measuring several properties related to the energy of the molecule during its entry and exit from the pore.
Critically, the goal is to measure whether the interaction between the molecule and its environment is primarily due to chemical bonding or the ability of the molecule to wiggle and move freely throughout the capture process and release.
Until now, reliable measurements to extract these energetic components have been lacking for a number of technical reasons. In the new study, a team co-led by Joseph Robertson of NIST and Joseph Reiner of VCU demonstrated the ability to measure these energies with a laser-based rapid heating method.
Measurements should be made at different temperatures and the laser heating system ensures that these temperature changes occur quickly and reproducibly. This allows researchers to perform measurements in less than 2 minutes, compared to the 30 minutes or more it would otherwise take.
“Without this new laser heating tool, our experience suggests that the measurements simply will not be made; they would take too long and cost too much, ”said Robertson. “Essentially, we have developed a tool that can change the nanopore sensor development pipeline to quickly reduce the guesswork involved in sensor discovery,” he added.
Once the energy measurements are taken, they can help reveal how a molecule interacts with the nanopore. Scientists can then use this information to determine the best strategies for detecting molecules.
For example, consider a molecule that interacts with the nanopore primarily through chemical interactions – primarily electrostatic. To achieve the Goldilocks capture time, the researchers experimented with modifying the nanopore so that its electrostatic attraction to the target molecule was neither too strong nor too weak.
With this goal in mind, the researchers demonstrated the method with two small peptides, short chains of compounds that form the building blocks of proteins. One of the peptides, angiotensin, stabilizes blood pressure. The other peptide, neurotensin, helps regulate dopamine, a neurotransmitter that influences mood and may also play a role in colorectal cancer. These molecules interact with nanopores mainly through electrostatic forces. The researchers inserted into the nanoparticles of gold nanopores coated with a charged material that stimulated electrostatic interactions with the molecules.
The team also looked at another molecule, polyethylene glycol, whose ability to move determines how long it spends in the nanopore. Ordinarily, this molecule can move, rotate and stretch freely, unencumbered by its surroundings. To increase the residence time of the molecule in the nanopore, the researchers altered the shape of the nanopore, making it more difficult for the molecule to squeeze through the tiny cavity and exit.
“We can exploit these changes to build a nanopore biosensor suitable for the detection of specific molecules,” says Robertson. Ultimately, a research lab could use such a biosensor to identify biological molecules of interest, or a doctor’s office could use the device to identify markers of disease.
“Our measurements provide a blueprint for how we can alter pore interactions, whether through geometry or chemistry, or a combination of the two, to tailor a nanopore sensor to detect specific molecules, count a small number. molecules, or both, “says Robertson.