In order to meet the
ever-growing demand from both industry and research-institutes for better,
faster and more reliable means of sensing and studying biological systems, the
division of Biological Physics aims to explore and develop novel surface-based analytical
methods and techniques. Our goal is to
provide better insight and understanding of how biological systems work and
interact with each other and simultaneously contributing in the fight against
some of the most demanding healthcare-issues in society, such as Alzheimer’s
decease and virus infections.
To achieve this aim the group
seeks to generate a cross-disciplinary working environment consisting of
engineers, chemist, physicists, biologist, mathematicians and other type of
scientist. Aided by latest achievements
and results within each field and in combination with high-end laboratory
facilities and computer simulations, these scientists form a powerful unit that
takes on some of the most demanding issues in quantitative biology and related
disciplines.
Evanescent-light
microscopy for label-free single molecule detection
The group has
been involved in developing a novel waveguide chip for evanescent-light
illumination of nanoscopic objects in aqueous environments.
Illumination-light
is coupled into the chip using a standard single-mode optical fiber. The light
travels through the chip generating a surface-bound light at the core-cladding
boundary. A sensing region is formed by
partially removing the upper part of the cladding layer, exposing the core
layer of the wave-guide to a solution containing the specimen to be
investigated. Objects within the range of the surface-bound light will interact
with it while objects outside it will be left unaffected. In this way, the signal-to-background ratio
from surface-immobilized objects is greatly enhanced, which can be especially
beneficiary when measurements have to be carried out in complex environments.
The generated signal, either scattered (green) or
fluorescent (orange), is then picked up by a standard
upright (or inverted) optical microscope.
Depending on the particular application, the
evanescent-light can be either single or multi-wavelength and made to extend
anywhere from 200 to 2000 nm into the solution containing the objects to be
monitored. Furthermore, the illumination
can be made to extend over macroscopic surface areas allowing for simultaneous
observation over a large field-of-view.
The device can be used for both labelled- and label free detection of
nanoscopic objects such as viruses, lipid vesicles or metallic nanoparticles.
It can also be used for monitoring near-surface area of larger biological
entities such as bacteria or cells.
Figure 1: 100 nm in diameter fluorescently labelled POPC lipid vesicles immobilised
on the surface of the chip observed though fluorescence and scattering
respectively.
Label-free
imaging of membrane heterogeneity
Surface-enhanced ellipsometry contrast (SEEC) imaging has been used for
time-resolved label-free visualisation of biomolecular recognition events on
spatially heterogeneous supported lipid bilayers (SLB). Using a conventional
inverted microscope, equipped with total evanescent-light illumination,
biomolecular binding events have been monitored with a lateral resolution close
to the optical diffraction limit with a sensitivity, in terms of surface
coverage, that is competitive with surface plasmon resonance (SPR) imaging and imaging ellipsometry (IE).
Figure 2:
SEEC micrograph showing supported
lipid bilayers with GalCer-rich domains
(dark) surrounded by POPC-rich surroundings (bright area).
Nanofluidic
with integrated plasmonics for label-free detection
Nanoscale sensors, such a plasmonic metal
nano-particles, can provide new ways of performing sensing that are not
possible with their large-scale analogues.
A small size sensing element can for example be used in combination with
a nanofluidic system allowing for efficient delivery of small analyses in low
concentration and low volume to the sensing surface.
The group has made efforts to fabricate and position nanoplasmonic
sensor elements within nanofluidic pores for fast and efficient label-free
detection. By producing arrays of pores
in a thin (220 nm) silicon nitride membrane with one plasmonic nanoparticle
sensor in each pore we
have developed a
high throughput polarization-sensitive plasmonic sensor, that can be tuned
significantly in the visible wavelength range by just varying one process parameter. This has been used to carry out label-free
measurements of changes in local refractive index
within the nanopores, thus paving the way for using the device for label-free plasmonic biosensing.
Figure 3:
Plasmonic structures integrated in nanofluidic pores for high-throughput
plasmonic sensing.
Single molecule
imaging and kinetics
Using total internal reflection fluorescence (TIRF) imaging of
fluorescently labelled lipid vesicles, (synthetically made or directly derived
from cell membranes) enables monitoring of single molecule binding events. This can help to reveal information about
binding kinetics of ligand interactions with cell membrane bound receptors.
Due to the single-molecule sensitivity of the system,
measurement and analysis are possible without the need for over-expressing
membrane proteins, which makes the assay especially attractive in early drug-
screening applications.
Figure 4: Single-molecule binding events are
detected with high spatial and temporal resolution by monitoring fluorescently
labeled lipid vesicles.
Selected References
1.
Gunnarsson, A., M. Bally, P. Jonsson, N. Medard and F.
Hook (2012). Time-resolved surface-enhanced ellipsometric contrast imaging
for label-free analysis of biomolecular recognition reactions on glycolipid
domains. Anal Chem 84(15): 6538-6545
2.
Gunnarsson, A., L. Dexlin, P. Wallin, S. Svedhem, P.
Jonsson, C. Wingren and F. Hook (2011). Kinetics of ligand binding to
membrane receptors from equilibrium fluctuation analysis of single binding
events. J Am Chem Soc 133(38): 14852-14855
3.
Mazzotta, F., F. Hook and M. P. Jonsson (2012). High
throughput fabrication of plasmonic nanostructures in nanofluidic pores for
biosensing applications. Nanotechnology 23(41): 415304
4.
Bally, M., M. Graule, F. Parra, G. Larson and F. Höök
(2013). A virus biosensor with single virus-particle sensitivity based on
fluorescent vesicle labels and equilibrium fluctuation analysis."Biointerphases
8(1): 4
5.
Bally, M., A. Gunnarsson, L. Svensson, G. Larson, V.
P. Zhdanov and F. Höök (2011). Interaction
of Single Viruslike Particles with Vesicles Containing Glycosphingolipids.”
Physical Review Letters 107(18)
6.
Kunze, A., M. Bally, F. Hook and G. Larson (2013). Equilibrium-fluctuation-analysis
of single liposome binding events reveals how cholesterol and Ca(2+) modulate
glycosphingolipid trans-interactions. Sci Rep 3: 1452.
Project members
Fredrik Höök (project leader)
Marta Bally
Anders Lundgren
Björn Agnarsson
Stephan Block
Björn Johansson
Mokhtar Mapar
Funding
European Metrology Research
Project (BioSurf)
Swedish Research Council
Swedish Foundation for
Strategic Research
Göran Gustafsson Foundation