Research

Ulrich F. Keyser et al.

Controlling molecular transport - DNA origami & solid-state nanopores - DNA nanopores in lipid membranes - Mimicking protein channels
Single molecule sensing - Transport through 2D materials - Transport through lipid membranes - Technology development

Our publications

Controlling molecular transport through nanopores




Single DNA molecules can be inserted and controlled in a single solid-state nanopore. The DNA is driven into the pore by an electrical field in the nanopore. We are able to stall and control the translocation by grabbing the end of the DNA strand using optical tweezers and study the influence of hydrodynamic interactions on the transport of DNA though these nanopores. We are investigating if it is possible to detect proteins bound to the DNA to determine the primary sequence. In addition, we work on extending the technique to single proteins in biological nanopores. In addition we discovered electrokinetic flow rectification and reversal in nanopores. Our measurements are important for the understanding of the whole transport process of the molecule.

Involved researchers:
Maria Ricci, Nick Bell

In collaboration with:
Sandip Ghosal (Northwestern University), Murugappan Muthukumar (University of Massachusetts, Amherst)

Key references:
N. Laohakunakorn, et al. Nano Letters, 13(6):2798-2802, 2013. [ DOI | http ]
O. Otto, S. Sturm, et al. Nature Communication, 4, 1780, 2013. [ DOI | .html ] N. Laohakunakorn, et al. Nano Letters, 15(1):695-702, 2015. [ DOI | http ]

DNA origami & solid-state nanopores




Videos:
DNA origami trapping movie
Recently, we demonstrated the assembly of functional hybrid nanopores for single molecule sensing by inserting DNA origami structures into solid-state nanopores. In our experiments, single artificial nanopores based on DNA origami are repeatedly inserted in and ejected from solid-state nanopores with diameters around 15 nm. We show that these hybrid nanopores can be employed for the detection of DNA molecules. Our approach paves the way for future development of adaptable single-molecule nanopore sensors based on the combination of solid-state nanopores and DNA self-assembly. Now we explore the possibilities to use custom-made DNA origami nanopores for mimicking protein nanopores as well as improve nanopore sensing and specificity and understand the ionic current transport through DNA origami derived systems. Recently, we reported the first comprehensive characterisation of the ionic conductivity of DNA origami plates using nanocapillary electric current recordings and all-atom molecular dynamics (MD) simulations. We show that increasing the concentration of Mg2+ ions makes the origami plates more compact, reducing their conductivity. An exciting future direction of this work is to explore the possibility of programming the electrical properties of a self-assembled nanoscale object using DNA.

Involved researchers:
Nick Bell, Elisa Hemmig, Silvia Hernandez-Ainsa, Jinglin Kong

In collaboration with:
Aleksei Aksimentiev (Urbana-Champaign), Tim Liedl (LMU Munich), Fernando Moreno-Herrero (CSIC Madrid), Oxford Nanopore Technologies

Reviews:
S. M. Hernandez-Ainsa, , and U. F. Keyser. Nanoscale, 6:14121-14132, 2014. [ DOI | http ]
N. A. W. Bell and U. F. Keyser. FEBS Letters, 588(29):3564-3570, 2014. [ DOI | http ]

Key references:
N. A. W. Bell, et al. Nano Letters (published 20.12.2011), 12(1):512-517, 2012. [ DOI | http ]
C.-Y. Li, E. A. Hemmig, et al. ACS nano, 9(2):1420-1433, 2015. [ DOI | http ]

DNA nanopores in lipid membranes




Videos:
DNA origami explanation
We recently presented self-assembled DNA nanostructures decorated with hydrophobic tags which form artificial transmembrane channels in lipid bilayers. Our synthetic nanopores resemble biological ion channels in dimensions as well as in behavior exhibiting gating and voltage-switching characteristics. Our approach paves the way for the development of more sophisticated man-made nanopores capable of mimicking the complexity of their natural counterparts. We are currently exploring new DNA-based nanopore designs and investigating structural properties to enhance their compatibility with lipid membranes. We are also exploiting the chemical and physical adaptability of DNA to fabricate smart channels capable of controlled response to external stimuli.

Involved researchers:
Silvia Hernandez-Ainsa, Kerstin Goepfrich, Alexander Ohmann, Elisa Hemmig

In collaboration with:
Aleksei Aksimentiev (Urbana-Champaign), Eugen Stulz (Southhampton), Mathias Winterhalter (JU Bremen), Stefan Howorka (UCL), Oxford Nanopore Technologies

Key references:
K. Goepfrich, T. Zettl, A. E. C. Meijering, S. Hernandez-Ainsa, S. Kocabey, T. Liedl, and U. F. Keyser. Nano Letters, 15(5):3134-3138, 2015. [ DOI | http ]
A. Seifert, K Goepfrich, J. Burns, N. Fertig, U. F. Keyser, and S. Howorka. ACS nano, 9(2):1117-1126, 2015. [ DOI | http ]
J. R. Burns, K. Goepfrich, J. W. Wood, V. V. Thacker, E. Stulz, U. F. Keyser, and S. Howorka.
Angewandte Chemie International Edition
, 52(46):12069-12072, 2013. [ DOI | http ]

Mimicking protein channels in microfluidic chips


Videos
Diffusing particles (4.5MB)
Tracking particles (2.8MB)
Non-decaying interactions - explained
Transport of ions, metabolite molecules and macromolecular solutes across biological membranes is an ubiquitous process in nature. Specifically membrane proteins form metabolite-specific channels with large aqueous pores exhibiting affinities to their metabolites. To understand the physics of molecular transport, we have developed a microfluidic model system that mimics protein membrane channels. This setup allows us to record trajectories of each particle, which are then used to characterise the Brownian motion, particle-channel interactions and particle-particle interactions. In addition, we mimic facilitated transport by creating synthetic binding sites with holographic optical tweezers. This offers a unique method for controlling the potential landscapes within the channels. Ultimately, we aim to understand how the channel-shape and -potential controls transport through them. Recently we characterised a novel mode of hydrodynamic interactions in narrow channels that mediate non-decaying interactions. We are now looking into systems where the particles diffuse in complex potential landscapes and are driven by externa forces.

Involved researchers:
Stefano Pagliara, Jannes Gladrow, Karolis Misiunas, Yizhou Tan

In collaboration with:
Stefano Pagliara (Exeter), Eric Lauga (DAMTP), Sergey Bezrukov (NIH, Bethesda), Miguel Rubi (Barcelona), Dirk Aarts (Oxford University), Rod Lim (Biozentrum, Basel)

Key references:
S. Pagliara, S. L. Dettmer, and U. F. Keyser. Phys. Rev. Lett., 113:048102, 2014. [ DOI | http ]
K. Misiunas, et al. Physical Review Letters, 115:038301, 2015. [ DOI | http ]

Single molecule sensing with glass nanopores




Videos:
DNA translocation through nanocapillary
Several translocations (Appl. Phys. Lett.)
We demonstrated the detection of the folding state of double-stranded DNA in nanocapillaries with the resistive pulse technique. This bench-top method allows for fabrication of nanocapillaries with diameters down to 10 nm. We studied translocation of DNA which is driven by an electrophoretic force through these glass nanopores. The resulting change in ionic current indicates the folding state of single DNA molecules. Our experiments prove that nanocapillaries are suitable for label-free analysis of DNA in aqueous solutions and viable alternatives to solid-state nanopores made by silicon nanotechnology. We extended the method to the detection of single proteins in solution and through a combination with DNA origami self-assembly we are now able to identify proteins from complex mixtures. At the moment we are aiming to extend the feasibility of our method to measure binding constants and ultra-low protein concentrations. These glass nanopores are also ideal model systems to study the physics of the transport process due to the absence of unspecific surface interactions. Combination with optical techniques allows for complementary studies of the translocation process by fluorescence microscopy and optical tweezers (see above).

Involved researchers:
Nick Bell, Kaikai Chen, Jinglin Kong

In collaboration with:
Raymond Bujdoso (Veterinary School, Cambridge), Tuomas Knowles (Chemistry, Cambridge), Murugrappan Muthukumar

Key references:
L. J. Steinbock, et al.Nano Letters, 10(7):2493-2497, 2010. [ DOI | http ]
W. Li, et al. ACS nano, 7(5):4129-4134, 2013. [ DOI | http ]
N. A. W. Bell and U. F. Keyser. JACS, 137(5):2035-2041, 2015. [ DOI | http ]

Ionic transport through 2D materials

We have developed a faster and more reliable way of measuring how molecules cross graphene membranes. As the thinnest possible membrane we're interested in how we can exploit its unique properties for medical diagnosis, protecting smartphone screens or filtering water. Although graphene is a single atom thick its structure prevents any molecules from passing through it. However defects in the graphene lattice such as missing atoms or grain boundaries can create openings through which individual molecules can pass. In our method we seal a sheet of graphene across the opening of a glass nanocapillary. Using electrodes in the capillary and the reservoir we can monitor the current as ions cross the membrane. This allows us to rapidly and reliably measure transport and combine this information with Raman Spectroscopy. By varying the conditions and molecular species we are now working to understand the physics under pinning molecular transport across 2D materials. In particular we are interested in the selectivity of different defects and methods to controllably induce defects. We will use this information to engineer nanoporous graphene membranes by tuning the defects using various different methods to remove atoms or close existing defects. Graphene is exciting an exciting membrane material because its atomic thickness promises performance that cannot be achieved with other membrane materials but working with such a thin membrane presents many challenges.
This project is a collaboration with the Hofmann group and funded by EPSRC Graphted EP/K016636/1.

Involved researchers:
Michael Walker, Mustafa Caglar

In collaboration with:
Stephan Hofmann (CAPE, Cambridge)

Key references:
M. I. Walker, et al. Appl. Phys. Lett., 106:023119, 2015. [ DOI | http ]
M. I. Walker, et al. Appl. Phys. Lett., 107:213104, 2015. [ DOI | http ]

Drug and molecular transport through lipid membranes


Passive membrane transport is ubiquitous in living organism. One class of special interest are small organic compounds like indole. In many respects indole behaves like the signalling component of a quorum sensing system. Indole synthesised within the producer bacterium is exported into the surroundings where its accumulation is detected by sensitive cells. By direct observation of indole import into individual liposomes we have shown that indole can cross a lipid membrane without the aid of a proteinaceous transporter and provide a simple explanation for the ability of indole to signal between biological Kingdoms.
Our microfluidic technique enables quantification of drug transport through lipid membranes. Since assive transport accounts for over 80% of drug uptake in cells it has special relevance for the understanding of antibiotic resistance. Giant unilamellar vesicles (GUVs) are used as model membranes; the lipid composition of these membranes is fully controlled. Our optofluidic assay directly measures the Permeability Coefficient of a drug crossing lipid membranes. We are able to screen the permeability properties of various autofluorescent drugs in a lipid specific manner, without needing to resort to octanol partition coefficient measurements. Furthermore, we can study the effect of membrane proteins on drug transport. We are now using our system to quantify drug transport in highly controlled environments with special emphasis on clarifying the role of outer membrane channels in antibiotics resistance.

Involved researchers:
Jehangir Cama, Sowmya Purushothaman, Kareem Al Nahas, Michael Schaich

In collaboration with:
Stefano Pagliara (Exeter), Mathias Winterhalter (JU Bremen), David Summers (Genetics, Cambridge), Fiona Gribble and Frank Reimann (Institute for Medical Research, Cambridge)

Key references:
C. Chimerel, et al. BBA - Biomembranes, 1818(7):1590-1594, 2012. [ DOI | http ]
J. Cama, et al. Lab Chip, 14:2303-2308, 2014. [ DOI | http ]
J. Cama, et al. JACS, published online, 2015. [ DOI | http ]

Technology development




Many aspects of our work are only possible through the development and improvement of novel and established techniques. Over the last few years we focussed on several single molecule and particle techniques. Using the DNA origami technique we could create nanoparticle dimers that can be used for surface enhanced Raman spectroscopy. Another example concerns the online tracking of particles in magnetic and optical tweezers. The use of video microscopy allows to greatly simplify force measurements with optical tweezers with sub-pN and nm resolution. Our experience with particle tracking allowed to study the diffusion of particles in molecular velcro made from proteins isolated from the nuclear pore complex. We developed a novel approach for the patch-clamping of proteo-liposomes that was largely inspired by the vesicle prep sold by Nanion. Our microfluidic technology enabled the discovery of auxetic nuclei in embryonic stem cells. In general, we are always lookimg for good (biological) questions, where we can develop a new technique for obtaining quantitative data.

Involved researchers:
whole group

In collaboration with:
Stefano Pagliara (Exeter), Jeremy Baumberg (Cambridge), Ralf Seidel (Leipzig), Kevin Chalut (SCI, Cambridge), Mathias Winterhalter (JU Bremen), and many more

Key references:
V.V. Thacker, L. O. Hermann, D. O. Sigle, T. Zhang, J. J. Baumberg, and U. F. Keyser. Nature communications, 5:3448, 2014. [ DOI | .html ]
A. Huhle, D. Klaue, H. Brutzer, D. Daldrop, O. Otto, U. F. Keyser, and R. Seidel. Nature Communications, 6:5885, 2015. [ DOI | .html ]
T. Gutsmann, T. Heimburg, U. F. Keyser, K. R. Mahendran, and M. Winterhalter. Nature Protocols, 10:188-198, 2015. [ DOI | .html ]
S. Pagliara, K. Franze, C. R. McClain, G. W. Wylde, C. L. Fisher, R. J. M. Franklin, A. J. Kabla, U. F. Keyser, and K. J. Chalut. Nature Materials, 13:638-644, 2014. [ DOI | .html ]
K. D. Schleicher, S. L. Dettmer, L. E. Kapinos, S. Pagliara, U. F. Keyser, S. Jeney, and R. Y. H. Lim. Nature Nanotechnology, 9:525-530, 2014. [ DOI | .html ]