Novel analytical techniques and methods for biosensing

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


European Metrology Research Project (BioSurf)
Swedish Research Council
Swedish Foundation for Strategic Research
Göran Gustafsson Foundation


Published: Mon 20 Oct 2014. Modified: Tue 20 Sep 2016