LABTips: Mitigating Common Errors in Particle Analysis

By Candace Kastanis, Science Writer

Used in many diverse industry applications, particle analysis (PA) is a methodology used in laboratory settings to isolate and characterize particulate matter. Common methodologies used in PA activities include the traditional sieve method, DIA (dynamic image analysis), laser diffraction, TEM (transmission electron microscopy), and DIS (dynamic light scattering). Each PA method tends to come with its own set of nuances. Modern methodologies such as DIS and TEM (transmission electron microscopy) offer some measurable advantages in predetermining particle size and details about its shape but demonstrate some limitations in certain application types.

Nanoparticle isolation in particular is surging in popularity because nanoparticles possess strategic attributes that assist in creating more efficient drug delivery systems in molecular drug development. However, nanoparticle characterization is known for problems in reproducibility. Below are some tips on optimizing sample preparation and avoiding common errors in specific particle analysis application types.

Consider Using Various Methodologies and Techniques for Isolating Nanoparticles in Whole Blood Samples

DLS or dynamic light scattering sometimes called quasi-elastic light scattering is a common technique that is ideal for determining size and measurements for smaller molecules (0.6 nm to 6 um), but it is not suited well for larger particles. In whole blood samples, the number of large particles (e.g., platelets ~ 1 um, red cells at ~7-8 um, and white blood cells at ~ 12 um) makes the range too difficult to identify the Individual contributions and, in many cases, the color is often too opaque to deliver optimal results. Alternatively, consider using plasma instead of whole blood for characterization purposes.

Plasma is free of large particles and cellular debris, and it allows researchers to gain more information about the smaller particles. Also in plasma, the clotting factors are intact and a crucial element in achieving particle surface interactions, including them in the assay is recommended.¹

For plasma, consider using these parameters for best results:

• Independent measurements of fluid viscosity will allow for a more accurate interpretation of the autocorrelation curves from the DLS rig.
• Using a direct thrombin inhibitor as an anticoagulant choice is recommended as using traditional anticoagulants such as heparin can become problematic. EDTA, ADC, or sodium citrate will bind divalent cations, thereby possibly distorting your conclusions based on the DLVO effects.
• Prepared plasma samples for use in a DLS system may be a bit too broad. An alternative approach may be to use NTA along with fluorescent tags to identify nano-proteins or other particulate matter.

Understanding Ionic Strength Crucial Element in Protein-specific Particle Analysis

Simple mistakes can occur when the ionic strength is not considered and can have a dramatic impact on the behavior of ions in solution.² Two strong considerations related to ionic strength and protein characterization are:

• Salting Out Proteins:  Because proteins are notoriously small and unstable (temperature and Ph sensitive), any target proteins are difficult to separate out from other noise proteins classified using spectrometry-based methods.³ Salting-out can achieve molecule purification but is not suitable for isolating a specific protein. However, you can utilize the salting-out method to categorize the proteins before moving toward the isolation phase.

• Reagent Selection:  Chaotropic reagents are known to increase the efficiency of extraction by breaking down protein structure, but they require high ionic strength. Consequently, the salt removal process may destroy the protein. There are a few different reagents that may be used alternatively and result in efficient extraction for further downstream processes without destruction to the protein analytes. Consider your instrument availability, sample size and protein type, and the project timeline as guideposts for reagent selection.⁴

Solvent Choices Can Work for or Against You in Certain Applications

Matching the molecular components of a solute perfectly in terms of a solvent choice can be tricky. There is not a one-size-fits-all type solvent since solvent selection is determined by what is known to work best in the intended application type. It is a widespread practice to take the path of least resistance and start with water. Though as a solvent, larger particle solubility in water tends to be slow and incomplete.

Subsequently, understanding how certain solvents react in common particle analysis techniques can help avoid issues in characterization. Particle sizing can become significantly more challenging when particles are introduced to unfavorable temperatures or solvents. Therefore, it is helpful to understand a few situations where certain solvent choices make all the difference – especially in the case of nanoparticle sizing and characterization. Here are a few examples:

• Best practices normally point to using a dry analysis method when analyzing metal powders. However, when the samples contain only a minute amount of the powder, it may prove beneficial to pivot to a wet dispersion system using a surfactant and ethanol or methanol combination as a solvent choice to avoid sedimentation and flocculation problems. Because silver nanoparticles are becoming so popular in viral drug therapies, this tip may come in handy. Before moving on to more powerful solvents, stirring the solute/solvent mixture may enhance solubility because of increased temperature and surface area of smaller particulates.⁵

• Acetone or Ethanol? Both solvents work well in most particle analysis applications with only a few caveats. Acetone may be more advantageous in the isolation of nanoparticles because it can separate from the solution easily because it reacts with vanillin in spectroscopy. However, because ethanol is a hydrocarbon and can dissolve both polar and non-polar molecules, it tends to be a more popular solvent choice. Acetone is also known to demonstrate size and shape alterations in silver nanoparticles following laser ablation. For ultimate decision-making, lean into polarity-based matching using physiochemical and morphological properties as a guidepost for determining which solvent may work best.⁶

References

1. Leeman M, Choi J, Hansson S, Storm MU, Nilsson L. Proteins and antibodies in serum, plasma, and whole blood-size characterization using asymmetrical flow field-flow fractionation (AF4). Anal Bioanal Chem. 2018 Aug;410(20):4867-4873. doi: 10.1007/s00216-018-1127-2. Epub 2018 May 29. PMID: 29808297; PMCID: PMC6061777.
2. Rasmussen, M.K., Pedersen, J.N. & Marie, R. Size and surface charge characterization of nanoparticles with a salt gradient. Nat Commun11, 2337 (2020). https://doi.org/10.1038/s41467-020-15889-3
3. Kim MS, Zhong J, Pandey A. Common errors in mass spectrometry-based analysis of post-translational modifications. Proteomics. 2016 Mar;16(5):700-14. doi: 10.1002/pmic.201500355. PMID: 26667783; PMCID: PMC5548100.
4. Ashraf Kharaz Y, Zamboulis D, Sanders K, Comerford E, Clegg P, Peffers M. Comparison between chaotropic and detergent-based sample preparation workflow in tendon for mass spectrometry analysis. Proteomics. 2017 Jul;17(13-14):1700018. doi: 10.1002/pmic.201700018. Epub 2017 Jun 22. PMID: 28547889; PMCID: PMC5575552.
5. Biswas A, Maloverjan M, Padari K, Abroi A, Rätsep M, Wärmländer SKTS, Jarvet J, Gräslund A, Kisand V, Lõhmus R, Pooga M. Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery. Pharmaceutics. 2023 Jan 24;15(2):396. doi: 10.3390/pharmaceutics15020396. PMID: 36839718; PMCID: PMC9963036.
6. Webb, C., Khadke, S., Schmidt, S. T., Roces, C. B., Forbes, N., Berrie, G., & Perrie, Y. (2019). The Impact of Solvent Selection: Strategies to Guide the Manufacturing of Liposomes Using Microfluidics. Pharmaceutics, 11(12). https://doi.org/10.3390/pharmaceutics11120653