Nano Discovery Inc.

Protein detection and analysis is an essential part of the life science and biomedical research. A daily task of biomolecular researchers is to study and analyze the function and activity of proteins in biological systems. The study of a protein is not just to determine its concentration, more importantly, we need to understand its interaction with other molecules in the biological system. A protein molecule must "socialize" with other biomolecules to play its biological role and functions. The elucidation of biomolecular interactions holds the key to the understanding of various cellular processes and disease mechanisms. Here, we highlight briefly a few unique applications of NanoDLSay™ for protein research. As you will see, all these applications are related to the "size" analysis capablity of NanoDLSay™. Please refer to APPLICATION NOTES for more technical details. All referred applications have been demonstrated in published work (LITERATURE).

I. Real-time monitoring of protein-protein interactions

• First immobilize one target protein (A) on the AuNPs
• Mix the binding protein (B) with the target A-conjugated AuNPs
• Incubate the solution and monitor the nanoparticle size change of the assay solution over incubation time
• Directly compare the relative binding affinity of different protein partner by conducting such studies under the same conditions
• Additional assay formats may be designed and used to probe the different binding sites and binding mechanisms of two proteins

II. Protein size analysis—distinguish target protein as a monomer, complex or aggregate

• Assay conditions can be designed to allow the binding between protein and AuNP to reach a saturated level
• At a saturated binding level, the net increase of the particle size is roughly equivalent to twice of the hydrodynamic diameter (D) of the target protein
• A protein complex will cause a larger particle size increase than a protein monomer
• The presence of a protein aggregate will cause a continuous and unusually large average particle size increase of the assay solution. It is difficult for the particle size to reach a stable level and the particle size polydispersity will increase substantially

III. Protein complex detection and binding partner screening

• First, “catch” the target protein using a specific AuNP immunoprobe
• Observe the particle size increase to determine if a protein complex is present
• Without isolating the assay product, directly add the specific antibody for the suspected binding partner, and continue to monitor the particle size change of the assay solution.
• If the addition of a specific antibody causes a further particle size increase of the assay solution, the suspected binding partner is present in the complex
• Control assays should be conducted carefully to validate the results

IV. Label-free detection of protein oligomers and aggregates in real biological samples

Protein oligomers and aggregates are associated with Alzheimer’s disease and prion disease. Indeed, protein oligomerization and aggregation is involved more widely  in the biological functions and processes of cells than has been previously recognized.  For example, it has been discovered recently by two independent research groups that protein aggregation is also associated with cancer (Huo Q. Colloids Surf B Biointerfaces 2010, 78, 259-265, and Xu J. et al. Nat Chem Biol 2011, 7, 285-295).

However, not many techniques are available to detect protein oligomers and aggregates directly from the biological samples and fluids.  The limited number of traditional techniques for protein aggregate detection, such as size exclusion chromatography and ultra-centrifugation, are in most cases only applicable to aggregate detection from pure protein solution, but not from real biological samples. Fluorescence detection techniques typically require the labeling of the target protein with a fluorescent probe, which significantly limits the application of this technique.

NanoDLSay™ provides the most sensitive and informative tool for protein aggregate and oligomer detection directly from real biological samples. There is no need to label the target protein. An AuNP immunoprobe is used to “catch” the specific target protein. If the protein exists in oligomers or aggregates, this will cause AuNP cluster formation, and subsequently, a substantial particle size increase of the assay solution. The use of AuNP as a light scattering probe ensures that only the target protein oligomers and aggregates are detected from the complex samples.