A team from the University of Oxford have developed a microchip-based method that can be used to quickly and precisely determine the size of a wide variety of molecules. Their study is published today in the journal Science.
All molecules experience the forces of friction as they move through a fluid, and this friction can vary from one molecule to another depending on its properties. Existing techniques for determining size and shape of molecules make use of this fact; for instance, larger molecules travel shorter distances than smaller ones, on average, over a given period of time.
Now, a team from Oxford Chemistry have improved upon these longstanding techniques by engineering a microchip that exploits the impact of confinement on molecular motion in order to more precisely determine molecular size and shape. Their simple benchtop experimental approach offers high-speed readouts of molecular properties, with a precision and resolution generally expected from heavy-duty techniques such as X-ray scattering.
Cylindrical pockets less than a micrometer in diameter and depth etched into the surface of the microchip’s channels can “trap” molecules as they move through a parallel-plate channel. Larger molecules, once confined, spend longer in the pockets than smaller molecules because it is harder for them to escape back into the narrow channel from where they came – think of threading a needle, which is more difficult with thick thread than with thin. This nanoscale confinement harnesses the effects of entropy, giving a huge amplification in response and a consequent increase in precision and accuracy compared to existing techniques.
The team observed the molecules’ motion in solution through the pocket-filled channels using fluorescence microscopy, and the escape time data they recorded allowed them to reverse-engineer useful molecular size and shape properties. While this relatively simple method does not offer an atomistic view of the structure of molecules, the coarse-grained yet precise data it generates can be used to address many relevant bioanalytical questions.
Their method has been used to measure molecular weights for a range of biological molecules from 500 Da to at least 500 kDa, and can detect differences in weight down to two carbon atoms or less for smaller molecules. It can also be used to model a molecule’s three-dimensional structure, and the team have shown its applicability in medical diagnosis by sensing insulin concentration in serum at the sub-nanolitre scale.
Prof Madhavi Krishnan, who led the study, said:
This technology is likely to benefit biomolecular analytics in the broadest sense. Our ability to count and characterise rare species and states holds particular promise for molecular diagnostics. Yet another completely orthogonal sphere of interest is the rapid yet precise determination of the shape and conformation of biological molecules.
The Oxford team worked in collaboration with researchers from the Technical University Dresden and the University of Southampton, who provided key biologically-significant molecular systems for testing.
A particular advantage of this new solution-based method is that it allows researchers to measure the properties of biological molecules in their native state – i.e., dissolved in water and freely diffusing in the absence of any applied forces – unlike many current methods which rely on strong external fields or attachment of the molecules to surfaces.
The full study, Single-molecule stereometry, can be read today in the journal Science.
