Several strategies have been applied for designing dual or multiplex functional nanoparticle-based biolabels for a variety of biological applications. However, many such nanoparticles are mainly applied for quantitative approaches and are rather limited in qualitative application . Use of bio-conjugated quantum dots for fluorescence monitoring and for live-cell imaging is a major advancement towards qualitative single molecule imaging . At the same time, several reports show that cellular and subcellular interaction with these biolabel results in unspecific pathway mechanism and non-physiological behavior. Some of the scenarios include multiple crosslinking of the bio-conjugated nanoparticle with the cell surface receptor resulting in limited cellular uptake and deviation from the route pathway . For instance, it has been reported that transferrin-functionalized nanoparticle mimic native transferrin cellular internalization but not the recycling pathway. Such internalized transferrin-functionalized nanoparticle traffic from early endosomes, late endosomes is finally accumulated in the lysosomes. By using fluorescent nanoparticle or quantum dots, effective subcellular tracking is possible upto the point when there is no dissociation of transferrin ligand from the nanoparticle. It is indeed a drawback when the target ligand dissociates from the tracking device (in this case it is fluorescent nanoparticle). Hence, the use of existing fluorescent nanoparticle-based subcellular tracking limits in understanding and deciphering native conditions in the cellular and subcellular context. For the first time, we propose a conceptual design of the use of biarsenical labeling combined with nanoparticle/quantum dots as nanoswitch for imaging and cell tracking. Using this novel strategy, we propose to establish a robust on or off system governed mainly by the target ligand association or dissociation.
New Insights and Conceptual Advances: Nanoswitch is an interesting domain of nanotechnology which has the capability to generate either ‘ON or OFF’ states with nanoscale in size. Depending on the kind of nanomaterials used as a switch, its application can range from IT, material science, healthcare and molecular biology. With respect to healthcare applications, majority of the nanoswitches are generally DNA based and thereby its applications are rather niche. DNA-nanoswitches are predominately with target substrate being limited to nucleic acids. Other applications are dependent on the type of dominant non-biological nanomaterial used for the nanoswitch design. At the same time, use of nanomaterials and biomolecules in combination for nanoswitches has not yet been explored to a larger extent. These hybrid systems are limited to preliminary applications such as quantitative platforms and gel-based analysis. Presently, several protein nanomaterials are being used for biophotonics and photoacoustic nanoswitch for confocal microscopy and in vivo sensing applications. However, such nanoswitches are limited due to specificity and selectively issues. For the first-time, we describe here the use of chemical molecules functionalized on the nanoparticles that recognize tagged biomolecules with fluorescence ON/OFF monitoring. Such nanoswitches relay on generic tag with target biomolecules being DNA/RNA, protein or living cell, there-by broadening its range of applications from diagnostics to in vivo live imaging to systems biology.
Biarsenical Probe: FlAsH and ReAsH—Tetracysteine-based detection technology.
Invented by Tsien RY and colleagues, this unique biochemical method uses Fluorescein arsenical helix/hairpin binder ((FIAsH-EDT2) and Resoruﬁn-based arsenical helix/hairpin binder (ReAsH- EDT2) with bis-1, 2-ethanedithiol (EDT2 adduct) which has specific covalent labeling that is recombinantly tagged to protein molecules or peptide. The mechanism of the labeling system relies on affinity-based efficient selective binding of FIAsH/ReAsH containing two arsenic atoms to appropriately arranged tetra-cysteine motif with two non-cysteine amino acids (X) in the middle (Cys-Cys-X-X-Cys-Cys). It is highly specific, with high signal to noise ratio and is membrane permeable. Moreover, it has a very small size occupancy of 25 times smaller compared to fluorescent protein like GFP or CFP that can be fused to the protein. Several reports show that protein functionality and locality in subcellular conditions is being perturbed due to the fusion of classical fluorescent protein. However, tagging of tetracysteine-motif with protein did not show any significant perturbation, especially with GPCR, c-AMP and beta-tubulin. Further FIAsH/ReAsH motif has been used for FRET analysis with fluorescence that can be acquired from GFP despite high dithiol competitor concentration. At the same time, these probes are not devoid of disadvantages in their applicability. Some of the disadvantages include photobleaching, stability of the governing selective amino acid (X) in the motif, and pH governed inference in the fluorescence. Here, we propose to use fluorescent nanoparticle/quantum dot along with the FIAsH to complement its properties and to limit the disadvantage of its nativity (Supplementary Figure 1). This would include use of FRET properties by FIAsH to limit photobleach, biocompatible functionalization of nanoparticle for improved pH stability and low background signal.