Single Nanoparticle Biosensor Imaging and Analysis Using Enhanced Darkfield Hyperspectral Microscopy

There are a growing number of commercial entities developing plasmonic nanoparticles as biosensors for a wide range of disease detection assays. Additionally, nanoparticle biosensor basic research is growing at a rapid pace. In the past decade, the number of Google Scholar references in this area has more than doubled to over 28,300 in 2022.

Disease diagnosis via protein-conjugated nanoparticles can provide a very high level of detection, not always available with traditional assays. An example of this type of work was conducted by D. Pan et al. in 2020 entitled Selective Naked-Eye Detection of SARS-CoV-2 Mediated by N Gene Targeted Antisense Oligonucleotide Capped Plasmonic Nanoparticles https://pubs.acs.org/doi/full/10.1021/acsnano.0c03822.?Here, single thiol-modified ASO-capped AuNPs biosensors agglomerate selectively in the presence of minute amounts of target RNA sequence of SARS-CoV-2, indicating the presence of the virus. The sensitivity of this test is much higher than most commonly available assays.?

Developing nanoparticles for effective biosensor disease detection applications requires imaging and diagnostic capabilities specifically designed for the task. Traditional brightfield or fluorescent microscopy techniques are simply not a viable option for imaging nanoparticles. Electron microscopy, while providing high-resolution images of nanoparticles, has significant limitations due to the complex nature of sample preparation, its inherent low throughput nature, and the general in-accessibility by most research groups to an electron microscope.??

To meet the imaging and analysis needs for nanoparticle biosensor development, CytoViva, Inc has developed Enhanced Darkfield Hyperspectral Microscopy. This imaging system has been demonstrated to accurately image and spectrally characterize nanoparticles in different environments, including aqueous, cell or tissue-based environments. The system utilizes patented enhanced darkfield microscopy optics, which create high signal-to-noise images. This enables the endogenous scatter from nanoparticles to be easily observed in a wide range of environments, without any required sample preparation. When combined with hyperspectral imaging, each nanoscale image pixel contains the VNIR 400nm-1,000nm optical spectral response of that pixel’s spatial area. This allows the nanoparticles to be spectrally characterized to confirm nuanced changes in the presence of DNA, RNA, or other related targets.

Moreover, the non-destructive CytoViva, Inc technique requires no labeling, special fixation, or other alteration of the sample prior to imaging. It can identify most types of nanomaterials in either stained or unstained tissue and can be easily operated right from your benchtop.

A simple example illustrating how nanoparticles can be imaged and spectrally analyzed with the CytoViva, Inc system is shown in the examples above. In this example, the system is used to illustrate the amount of single nanoparticles vs aggregates in the displayed field of view. In Figure 1, an enhanced darkfield hyperspectral image of 50nm gold nanoparticles is shown at 240x total magnification. These spherical 50nm particles are in an aqueous solution, and the scatter of the single particles appear green and are known to produce a peak reflectance spectrum at 560nm.

Aggregates of AuNPs will red-shift, and those appear as yellow or red in the image. In Figure 2, differences are shown between the spectral response of a single 50nm particle (circled in green in Figure 1) and an aggregate of nanoparticles (circled in yellow). Note the 560nm peak of the single particles and an approximately 28nm red shift of the aggregates.

Figure 3 illustrates the spectral mapping of all single 50nm particles in the field of view.

In Figure 4, a class distribution report outlines the number of single particles vs aggregates in the sample image.

If your laboratory is developing nanoparticles as biosensors, enhanced darkfield hyperspectral microscopy can serve as a primary tool supporting the proper synthesis and protein functionalization of nanoparticles. It can also be used to determine changes in nanoparticles based on the presence of a biological target. The system can also be used to determine the uptake of nanoparticles by a biological target on both an in-vitro or ex-vivo basis. Please contact CytoViva at [email protected] to learn more or to discuss test imaging of your samples.???

Visit the?Schaefer Technologie GmbH?Website, Learn More about Different Products from CytoViva and Book a Demo Today:?CytoViva | Nanoparticles | Schaefer Group (schaefer-tec.com).