Solid-state nanopores for probing DNA and protein

Solid-state nanopores are small nanometer-scale holes in thin membranes. When used to separate two chambers containing salt solution, any biomolecule passing from one chamber to the other is forced to pass through the pore constriction. An electric field applied across the membrane is used to create an ionic current and electrophoretically drive charged molecules through the pore. As a molecule translocates through the pore, it causes a temporary reduction in the ionic current which is measured with a low-noise amplifier. The current blockade is a unique signature containing information about the volume and length of the molecule. The simple but versatile nanopore technique can be applied and modified in many ways, revealing a wide variety of phenomena. This thesis highlights some potential applications including the use of nanopores to determine polymer properties, the combination of DNA-origami nanostructures and nanopores, the use of nanopores to study knotting phenomenon in long polymers, and detection of DNA-bound protein. 'Solid-state nanopores' (vaste-stof nanogaatjes) zijn gaatjes van enkele nanometers in een dun membraan van bijvoorbeeld silicium nitride. Zo'n membraan met een nanogaatjes kan worden gebruikt om twee compartimenten met een zoutoplossing te scheiden. Wanneer er biomoleculen in de oplossing zitten, kunnen deze zich via het gaatje van het ene compartiment naar het andere bewegen. Als men een elektrisch veld over het membraan aanlegt, wordt er een ionenstroom gegenereerd en worden geladen moleculen via elektroforese door het nanogaatje gedreven. Een molecuul dat door het gaatje beweegt resulteert dan in een tijdelijke reductie van de ionenstroom, die wordt gemeten met een lage-ruis versterker. Deze tijdelijke stroomafname bevat unieke informatie over het volume en de lengte van het molecuul. De simpele en veelzijdige 'nanopore' techniek kan op veel verschillende manieren worden toegepast en gemodificeerd, waarmee een breed scala aan fenomenen kan worden onthuld. Dit proefschrift belicht een aantal mogelijke toepassingen, in het bijzonder wat betreft het gebruik van nanogaatjes voor het bepalen van polymeereigenschappen, de combinatie van DNA origami nanostructuren en nanogaatjes, het gebruik van nanogaatjes voor het bestuderen van knopen in lange polymeren en de detectie van DNA gebonden eiwitten.BioNanoscienceApplied Science

A nanopore sensor and method for selective detection of analytes in a sample

BN/BionanoscienceApplied Science

DNA nanopore translocation in glutamate solutions

Nanopore experiments have traditionally been carried out with chloride-based solutions. Here we introduce silver/silver-glutamate-based electrochemistry as an alternative, and study the viscosity, conductivity, and nanopore translocation characteristics of potassium-, sodium-, and lithium-glutamate solutions. We show that it has a linear response at typical voltages and can be used to detect DNA translocations through a nanopore. The glutamate anion also acts as a redox-capable thickening agent, with high-viscosity solutions capable of slowing down the DNA translocation process by up to 11 times, with a corresponding 7 time reduction in signal. These results demonstrate that glutamate can replace chloride as the primary anion in nanopore resistive pulse sensing.BionanoscienceApplied Science

Direct observation of DNA knots using a solid-state nanopore

Long DNA molecules can self-entangle into knots. Experimental techniques for observing such DNA knots (primarily gel electrophoresis) are limited to bulk methods and circular molecules below 10 kilobase pairs in length. Here, we show that solid-state nanopores can be used to directly observe individual knots in both linear and circular single DNA molecules of arbitrary length. The DNA knots are observed as short spikes in the nanopore current traces of the traversing DNA molecules and their detection is dependent on a sufficiently high measurement resolution, which can be achieved using high-concentration LiCl buffers. We study the percentage of molecules with knots for DNA molecules of up to 166 kilobase pairs in length and find that the knotting occurrence rises with the length of the DNA molecule, consistent with a constant knotting probability per unit length. Our experimental data compare favourably with previous simulation-based predictions for long polymers. From the translocation time of the knot through the nanopore, we estimate that the majority of the DNA knots are tight, with remarkably small sizes below 100 nm. In the case of linear molecules, we also observe that knots are able to slide out on application of high driving forces (voltage).Accepted Author ManuscriptBN/Cees Dekker LabBN/Technici en AnalistenEducation and Student Affair