True Orthogonality

Filling critical gaps in your nanoparticle characterization

Accurate nanoparticle characterization is critical across many fields. Whether you’re developing a new drug delivery system or ensuring the quality of commercial products, trustworthy particle measurements are the foundation for all downstream analyses. While optical methods like Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA) and flow cytometry (FC) are commonly used in the life sciences, researchers are increasingly understanding the limitations of these methods when it comes to measuring particles at the nanoscale.  These limitations become especially clear when analyzing complex systems like lipid nanoparticles and extracellular vesicles.

Optical techniques certainly have their place, but they struggle to provide the comprehensive insight needed for complex nanoparticle systems. This is where an orthogonal method becomes invaluable, helping you cross-validate results and gain a richer understanding. While high-resolution methods like CryoTEM exist, their slow speed and high cost make them unsuitable for most production or research settings. The nanomedicine industry, with its rapid growth and proliferation of diverse approaches to formulation, now has an urgent need for a simple and fast characterization solution that is truly orthogonal to the optical techniques.

When seeing falls short

Optical methods of nanoparticle characterization, specifically in biomedical applications, have two major pitfalls: Reliance on an optical detection method for heterogeneous particles with varying optical properties, and utilizing ensemble techniques of detection that fail to resolve polydisperse samples accurately. They also have inherent limitations from dependence on the effect of Brownian motion for detection, which can vary widely between samples and require time-consuming calibration steps to ensure accuracy. Below is an overview of each technique, along with an actual sample measurement performed with each of the respective methods (Polystyrene Control Beads: 52, 94, 122, and150 nm).

As you’ve seen above, reporting the real particle distribution is a challenge for both NTA and DLS. Since these methods do not collect and analyze data from single particles truly independently, they often present distributions as simplified, Gaussian-like peaks that do not reflect your sample’s actual complexity. While this kind of rough measurement can be sufficient in some cases, the advancement of nanomedicine and therapeutics urgently requires more accurate characterization to perfect formulations and maintain production consistency.

Microfluidic Resistive Pulse Sensing (MRPS) is a nanoscale adaptation of the Coulter Counter, which was patented in 1953 and has been widely adopted as a gold standard for analyzing micron-sized particles in clinical hematology and diagnostics. Patented and commercially available since 2014, Spectradyne’s MRPS delivers direct and accurate measurements of particle size and concentration deep into the nanoscale, even in challenging, complex samples.

The MRPS method is entirely non-optical—it does not use light scattering to detect or size particles.  Instead, it measures particle size by detecting an electrical signal (the pulse) generated when a particle displaces its own volume of conductive buffer as it passes through the sensing constriction.

Because MRPS is an electrical method that does not rely on light scattering to detect or size particles, it is truly orthogonal to the common optical methods like DLS, NTA and flow cytometry.  MRPS can therefore be used as an independent validation tool, and to deliver new insights that yield a more comprehensive understanding of your sample.

Beyond its value as a truly orthogonal technique, MRPS provides particle sizing accuracy equivalent to CryoTEM, one of the most highly trusted nanoparticle characterization methods. In a collaboration with NanoImaging Services, we analyzed a Lipid Nanoparticle sample using both MRPS and CryoTEM. The results, shown below, demonstrate that both measurements yield high resolution particle size distributions for the sample and lie in near-perfect agreement, with the additional advantage that MRPS measures absolute particle concentration.

Get the full application note here

MRPS delivers the most direct measurements of size & concentration at the nanoscale—True particle sizing that is independent of sample polydispersity or refractive index.  Read the original Nature Nanotechnology publication here.

MRPS alone is powerful, but what if you want to characterize a specific subpopulation of particles in your sample? Or quantify the fraction of your particles that have successfully encapsulated a payload?  Or measure the density of targeting ligand presented on the surface of some of the particles?

A new light: Fluorescence (F-MRPS)

In MRPS, each particle flows through a nanoscale channel that constrains its position very precisely.  That characteristic provided a unique opportunity to supplement MRPS with the power of fluorescence-based flow cytometry, without the optical alignment complexity inherent to conventional flow instruments. Leveraging microfluidics we could systematically—and automatically—align a laser to the nanoscale sensing channel and collect the emitted fluorescence light from each particle. The development of this technology culminated in the ARC particle analyzer, which combines all of the above advantages of MRPS with fluorescence detection on a single-particle level.

The two different techniques uniquely combined in Fluorescence-MRPS not only provide the high-accuracy particle size distribution analysis of MRPS, but also simultaneously enables a wide variety of fluorescence analyses with single-particle resolution. Ultimately, F-MRPS users quickly and easily measure encapsulation efficiency, surface ligand density, biological phenotype, viral titer, and more, directly in complex biological samples.

Fill in the blanks, and then some

Despite their lack of accuracy, optical measurement techniques like DLS can serve a useful role as a shared language among scientists.  But current developments in biomedical research increasingly require more informative particle characterization solutions that can keep up with production demands. F-MRPS and MRPS are rapidly filling that role, providing a solid foundation for research and development while also enabling precise quality control at-scale.

If your current protocols include only optical techniques for particle characterization, you are likely missing key insights into your samples that could make the difference between a successful formulation and wasted time and budget. To learn what F-MRPS and MRPS can reveal about your formulation, send us a sample for a free analysis today!

F-MRPS – Fast & easy to use