The research, published as an early view article in the journal Analytical Chemistry used nanoparticle optical biosensors conjugated to Immunoglobulin G (IgG) to detect and track the activity of individual cell receptors for a period of hours.
Activation of these receptors initiates the complex communication system within a cell, coordinating cell development, tissue repair and immunity responses.
Confocal microscopy is often used to image proteins on the surface of living cells, however, according to the researchers; the fluorescence probes used often suffer photobleaching.
This leads to a need for large numbers of fluorescence probes to be attached to antibodies in order for individual receptors to be detected and their binding mechanisms to be studied for extended lengths of time.
While attaching numerous probes to an antibody may seem like an obvious answer, only limited numbers of probes can be attached to antibodies and the more of these attached, the more likely they are to perturb the binding of the antibody to the protein receptor.
To overcome this problem, the researchers developed a non-photobleaching silver nanoparticle biosensor that enabled them to quantitatively measure the binding kinetics and affinity of single protein molecules on single living cells over several hours.
"The primary challenge of preparing effective nanoparticle biosensors, especially by covalent conjugation of protein molecules with nanoparticles, is to retain the biological activity of the biomolecules," wrote the authors.
The researchers achieved this and successfully imaged cells with low coverage of the receptor molecules and could distinguish bound biosensors by their slow diffusion on the cell membrane, which is orders of magnitude slower than the Brownian motion of unbound nanoparticles.
In addition, the authors noticed a significant red-shift of the response of the particles, which they attribute to a change of the surface properties and dielectric constant of the nanoparticles.
The researchers are now working on using the technique to study single ligand (hIL-2) and receptor molecules (hIL-2R) on living cells to enable the characterisation of anticancer vaccines and better understand their biological functions.
