A highly sensitive and selective diagnostic assay based on virus nanoparticles
One often overlooked area in designing biosensors, POC devices or IVD platforms is the protein immobilization. All these applications require proteins to be immobilized at the solid surface that are then used for detection of protein biomarkers, drugs of abuse, small molecules biological agents you name it. Proteins are however difficult beast to be tamed or captured. Proteins need their intact three dimensional structure to function at their optimum capacity and any small perturbation in their structure can severely impact their function. Traditional capture techniques rely in passive adsorption on variety of surfaces like nitrocellulose, polystyrene.Proteins go to the surface in any random orientation and once on the surface further interact with the surface that can lead to complete denaturation of the protein. By one estimate less than 10% of the antibodies adsorbed at the surface of ELISA plate is active for binding theor respective antigens. Same is the case when proteins are covalently captured using naturally occurring amines (lysines ) at the protein surface. Protein fusion tags that are typically used for protein expression and purification have also been used for oriented capture of proteins. Site specific modification of proteins for oriented capture is another clever approach to get maximum activity out of these tethered proteins. Options with antibodies are however limited.
With antibodies, adding a long chain linker or using carbohydrates for capture is most common. Now Jeewon Lee and colleagues have shown (Nature Nanotechnology 4, 259 – 264 (2009)) that by taming antibodies to orient them in specific orientation along with increasing the antibody density can lead to a million fold improvement over the conventional immunoassays. They combined nanoparticles made from virus proteins with proteins A to make small nanosized spheres that can grab antibodies in oriented fashion at very high density. They also added His tags to allow these spheres to be laid down on the surface. Using combination of these three innovations they were able to detect troponin 1 at attomolar level. This level of sensitivity is million fold better then commercial ELISA based kits.
This approach clearly shows that improving protein immobilization methods can dramatically enhance the performance of existing biosensors, POC and IVDs.