Characterisation of cell surface antigens and their interaction partners is one of the hot topics in scientific research and development. The commonly applied biochemical approaches have certain limitations which might be overcome by label-free technologies.

Conventional biochemical approaches

The understanding of cell-cell and cell-protein interactions does not only elucidate molecular mechanisms in basic research but also provides important insights into drug targets during the development of new pharmaceuticals. As a consequence, the demand for novel innovative cell-based assays is high.

There are plenty of different biochemical techniques available on the market to investigate biomolecular interactions. Many of them that are commonly used are so called end-point assays. After allowing the reagents to equilibrate, only one measuring point is taken in those techniques. Well-known examples are the immunoprecipitation/pull-down assay, the Western blot and the enzyme linked immunosorbent assay (ELISA). These techniques allow to identify direct binding partners and the subsequent cellular reaction after binding. However, a real-time monitoring and a kinetic evaluation of the binding is not feasible.

In contrast, label-dependent technologies like fluorescence live cell imaging and Förster resonance energy transfer (FRET)/bioluminescence resonance energy transfer (BRET) allow the visualisation of a molecular co-localisation in real-time, e.g. by fluorescence microscopy. However, those techniques require time-consuming and/or costly labelling steps with reporter proteins, which could even potentially disturb the functionality of the target molecule or the physiological microenvironment of a cell and therefore mask the nature of the interaction.

Label-free technologies

The great advantage of label-free technologies is of course that they are not based on labelling of biomolecules. Already widely applied in the last decades to study biomolecular interactions, label-free biosensors became recently more and more attractive for the investigation of whole cell interactions. The most prominent technologies which offer this possibility are based on either optical, electrochemical and piezoelectric approaches. This article will briefly present a selection of optical label-free approaches. The most striking beneficial hallmarks of these techniques are:

  • Acquisition of real-time data
  • High throughput measurements
  • Usually kinetic quantification possible

Optical label-free approaches rely on the detection of a biomolecular interaction on the surface of a specific transducer. Hence, also the binding of a cell to an immobilised ligand on this surface can be observed. Alternatively, cells themselves can easily be cultivated on transducers when coated with a thin layer of extracellular matrix (ECM) components (e.g. fibronectin, collagen). This allows the direct observation of the binding of a molecule to the cells as well as the subsequent whole cell reaction.

Reflectometric interference spectroscopy and single colour reflectometry

The reflectometric interference spectroscopy (RIfS), the single colour reflectometry (SCORE) and certain other optical approaches have all in common that their biosensor techniques make use of the interference of light. In RIfS and SCORE, biomolecules can be attached as ligands to the optical transducer through chemical functionalisation of a glass surface. Binding of an analyte alters the physical characteristics of the sensitive layer, which causes a detectable change in the interference of the reflected light. While RIfS relies on the implementation of white light, the SCORE technology uses monochromatic light that no longer requires the detection of a complete spectrum. This enables high-throughput kinetic measurements in an array format with up to 22,500 spots per measurement (1).


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Surface plasmon resonance

The surface plasmon resonance (SPR) is an evanescent field-based technology, which commonly comprises a noble metal layer on top of a glass prism as optical transducer. When coated with a biopolymer, the noble metal film facing the aqueous phase is able to carry biomolecules and molecular interactions can be monitored. In the special case of cell-based assays, these kind of techniques have certain limitations. In order to achieve cultivation of cells on an SPR biosensor, the gold layer nessesarily has to be further coated with e.g. ECM proteins. Because the depth of penetration of an evanescent wave is rather limited to around a few hundred nanometres from the noble metal layer on, the direct proof of a substance binding itself to cultivated cells is not feasible. Nevertheless, SPR biosensors enable the detection of whole cell intracellular downstream reactions or also binding of cells in suspension to an immobilised biomolecule (2).

Resonant mirror

Similar to SPR, resonant mirror biosensors are based on an evanescent field. Therefore, comparable considerations especially regarding the penetration depth and hence the detection radius have to be taken into account. Instead of a noble metal, a layer of a high refractive index medium is implemented. Because of the special setup of the light path, resonant mirrors display an enhanced sensitivity compared to SPR biosensors (3,4).

Photonic crystal

Photonic crystals are materials, which are composed of a periodic variation in the refractive index in the order of a wavelength. Because of this special architecture, molecular and cellular binding on such biosensor surfaces can be detected. Photonic crystals are available being incorporated into standard 96- and 384-well microplate formats. Therefore, cell-based assays using this kind of biosensors are easy to handle and relatively low cell numbers are sufficient for detection (5,6).

Raman spectroscopy

Raman spectroscopy relies on inelastic scattering of monochromatic light and gives conclusion about the actual structure of molecules and functional groups within a sample. A so called Raman spectrum of a live cell is able to deliver information about the total biochemical constitution of the cell including major biomolecules like DNA, lipids and proteins. Hence, the molecular consequences of a biomolecular binding to a cell can be measured as a change in the Raman spectrum, like e.g. the degradation of DNA after treatment of the cell with a specific toxin (7).

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Label-free: the new gold standard?

Label-free technologies comprise many remarkable advantages like high throughput, real-time monitoring and the ability to determine kinetic parameters of a biomolecular binding.

Since no labelling is required, the natural state of the biomolecule is preserved. Accordingly, label-free approaches indeed represent a powerful tool in the investigation of biomolecular interactions and the detection of cell responses in both basic research and pharmacological screenings. As any available interaction-detecting technique, they have to face certain limitations. Label-free approaches are generally less sensitive than label-based technologies. Furthermore, specific cellular reactions subsequent to the molecular interaction are difficult to discriminate, like for example the activation of a discrete downstream signalling pathway. Therefore, label-free techniques might not be suitable as stand-alone technology for all cell-based scientific issues. A combination of label-free and label-based approaches is recommended, utilising the advantages and power of both strategies in order to achieve comprehensive and reliable data.


Do you already combine cell assays and label-free approaches? Or are you interested in a label-free technique for your particular application?

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(1) Verzijl, D., et al. (2017) A novel label-free cell-based assay technology using biolayer interferometry. Biosens Bioelectron. 87: 388-395.
(2) Hide, M., et al. (2002) Real-time analysis of ligand-induced cell surface and intracellular reactions of living mast cells using a surface plasmon resonance-based biosensor. Anal Biochem. 302: 28-37.
(3) Watts, H.J., et al. (1994) Optical biosensor for monitoring microbial cells. Anal Chem. 66: 2465-2470.
(4) Daghestani, H.N. and Day, B.W. (2010) Theory and Applications of Surface Plasmon Resonance, Resonant Mirror, Resonant Waveguide Grating, and Dual Polarization Interferometry Biosensors. Sensors. 10: 9630-9646.
(5) Heeres, J.T. and Hergenrother, P.J. (2011) High-throughput screening for modulators of protein–protein interactions: use of photonic crystal biosensors and complementary technologies. Chem Soc Rev. 40: 4398-4410.
(6) Shamah, S.M. and Cunningham, B.T. (2011) Label-free cell-based assays using photonic crystal optical biosensors. Analyst. 136: 1090-1102.
(7) Notingher, I. (2007) Raman spectroscopy cell-based biosensors. Sensors. 7: 1343-1358.

This article was written by
Alice works as scientist and project manager at Biametrics. She has a Master in Molecular Medicine and experience in molecular biology of eukaryotic cells.