Label-free detection technologies may suffer from non-specific binding of irrelevant proteins within the sample. Functionalisation of the sensor surface with specific (bio)polymers can inhibit this non-specific binding to a minimum.
Interactions at the heterogeneous phase
Within this article, we review the functionalisation of solid sensor surfaces with biopolymers to block non-specific binding of irrelevant proteins. Often, experiments are performed in crude samples like blood, serum, lysates or other complex matrices. Here, plenty of different constituent parts are present, especially proteins. However, only one or a few of these proteins are the true analyte for the measurement, whereas the bulk is not of relevance for the experiment.
A special case: label-free methods
Label-free technologies offer the advantage that they do not rely on labelling the analyte with specific dyes (fluorescence) or radioactive isotopes (radiolabelling). These technologies detect the change of surface properties like physical thickness of layers or the surface-near refractive index or combinations of both. The binding of (bio)molecules to such sensor surfaces leads to a binding signal.
Non-specific binding: a nuisance
At first glance, the binding of all kind of molecules in label-free sensor systems leads to a binding signal, no matter if specific or non-specific. This may disturb the evaluation of the binding experiments. Label-free experiments are commonly performed on solid substrates (like glass or plastics) that are hydrophobic by nature. Unfortunately, proteins particularly bind to hydrophobic interfaces and then denature, which additionally increases their binding to the surface.
Besides the fact that non-specific binding of irrelevant proteins also leads to a signal and thus may falsify the results obtained from a binding experiment, non-specific binding can also lead to a blocking of binding sites located on the surface. Thus, the analyte within the sample can be hindered to interact with its respective binding site or partner on the surface, which also may lead to unsatisfying results.
How to eliminate non-specific binding
To distinguish specific from non-specific binding in label-free technologies, two approaches can be pursued separately or in combination:
- internal referencing (mathematical elimination of non-specific binding)
- inhibiting non-specific binding using surface chemistry (bio-chemical elimination of non-specific binding)
Applying surface chemistries to sensor surfaces may be interpreted as a disadvantage of these technologies. However, as the user wants to analyse specific (bio)molecular interactions between two specific (bio)molecules, these sensors have to be functionalised and treated with different bio-chemicals in any case in order to immobilise one of the two interaction partners. Also, biopolymers commonly provide beneficial properties for the immobilised biomolecules in terms of stability and activity.
Functionalisation of the surface with a suitable biopolymer is mandatory in label-free applilcations.
Which properties do biopolymers need to provide effective shielding of the sensor surface?
In order to effectively shield the sensor surfaces from non-specific binding, surface-bound biopolymers need to provide two essential characteristics:
- they need to be hydrophilic and highly hydratable
- their polymer chains need to be flexible
A surface-near hydration layer of tightly bound water molecules provides an energetic and physical barrier to prevent adsorption of proteins to the surface. The water molecules within this layer can effectively be bound either by hydrogen bonds (hydrophilic biopolymers) or via electrostatic interactions (ionic biopolymers) or both. When proteins would bind to such kind of layers, the tightly bound water molecules near the surface as well as the water molecules within the hydrate shells of the proteins would need to be expulsed, which results in an unfavourable reduction of entropy in these systems. The strength of surface hydration is mainly determined by the physicochemical properties of the used biopolymers (molecular weight, used surface chemistry) as well as the packing of their polymer chains on the surface (packing density, polymer chain conformation, film thickness).
Besides the degree of hydration of such biopolymers, their polymer chain flexibility is of great importance for their shielding properties. This is especially true for long-chain polymers. When proteins would bind to the surface, the polymer chains of the biopolymer would need to be compressed, which leads to steric hindrance and repulsion and thus to an unfavourable decrease of entropy.
Best protein resisting results can be obtained when the biopolymer provides both properties: strong surface hydration and highly flexible polymer chains.
Suitable biopolymers: a selection
The following selection of biopolymers makes no claim to be exhaustive. However, we would like to highlight some of the most commonly used biopolymer materials in label-free sensor technologies. This might be of interest especially if you are new to label-free sensor applications.
(Download the high-resolution table here.)
Which kind of biopolymers do you use for your experiments?
(1) Chen, L. Li et al. (2010) Surface hydration: Principles and applications toward low-fouling/nonfouling biomaterials. Polymer 51: 5283-5293.
(2) Zhang, H. et al. (2015) Anti-fouling Coatings of Poly(dimethylsiloxane) Devices for Biological and Biomedical Applications. J. Med. Biol. Eng. 35: 143-155.
(3) Statz, A. R. et al. (2005) New Peptidomimetic Polymers for Antifouling Surfaces. J. Am. Chem. Soc. 127: 7972-7973.