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IVD Technology Magazine
IVDT Article Index

Originally Published July 2000

Measuring the hydrodynamic radius of nanoparticle formulations

A dynamic laser light-scattering device provides rapid analysis of particles and aggregates for quality control in diagnostic test kits.

Many diagnostic test kits under development consist principally of monoclonal or polyclonal antibodies, or other large protein-based materials, coupled to a nanoparticulate carrier. A typical carrier is a gold particle less than 100 nm in diameter. The nanoparticles very often can aggregate as a function of acidity level, ionic strength, temperature, and concentration. Such test kits must be thoroughly characterized prior to filing with the regulatory authorities and should be batch tested for routine QA/QC purposes. The traditional analytical method for characterizing them has been electron microscopy, a technology that often requires hours or even days to provide accurate results.

Diagnostic test kits for pregnancy, food testing, and other purposes often are formulated as freestanding dipsticks or as devices enclosed in plastic housings. These products require several hundred microliters of sample blood, urine, food, or other material to perform the test. Certain size and surface characteristics of these nanoparticle formulations directly affect not only the way their surface chemistry interacts with the physiological specimen but also visualization of the test result by the clinician or patient. Manufacturing issues pertaining to membrane-based gold particle tests have been discussed recently in this magazine.¹ A clear understanding of these size, function, and stability relationships is vitally important for the development and process control of new diagnostic test kits.

Rayleigh or static laser light-scattering detectors have been used for more than a decade to determine the molecular weight characteristics of industrial synthetic polymers and biopolymers.² Recent innovations in modern high-speed electronic components such as high-performance diode lasers, high-speed digital signal processors, and avalanche photodiode detectors have fostered the development of a high-performance dynamic laser light-scattering system for the characterization of submicron or nanometer particle sizes for particles in solution, the PDDLS/Batch molecular size analysis system (Precision Detectors Inc.; Franklin, MA). This instrument and its associated software provide the following (see Figure 1).

• Measurements of hydrodynamic radius (R_h) within the range of 1.0 to 1000 nm.
• Deconvolution of molecular- and particle-size distributions in solutions and suspensions.
• High-sensitivity aggregation and self-association analysis of particles, antibodies, proteins, and colloidal or liposomal nanoparticle structures.

Light-Scattering Technology

When a polarized, monochromatic laser beam passes through a solvent containing biomolecules or nanoparticles, the amount of light scattered by these materials at an angle to the incident beam divided by the amount scattered by the solvent alone is directly proportional to the molecular weight of the molecule or particle multiplied by its concentration in the solution. This is called static light scattering. The technique has been known and used for decades in high-performance liquid chromatography and size-exclusion chromatography (HPLC/SEC) systems as a detector for determining the absolute molecular weight of eluting biomolecules.³

Figure 1. The PDDLS/Batch molecular size analysis system measures R_h and R_h distributions for biomolecules and nanoparticles in the range of 1.0 to 1000 nm. The cuvette-based unit requires only 100 µl of sample.

Proteins and nanoparticles in suspension also undergo Brownian motion that is related to their hydrodynamic radius as expressed by the Stokes-Einstein equation. As biomolecules or a distribution of biomolecules diffuse around the laser-beam coherence area, light scattered from them overlaps and interferes with the transmission of the laser light. A high-sensitivity detector can then record the time-varying signal caused by scattered light and compare it to the constant signal emitted when no molecules are present. This process is known as dynamic light scattering (DLS), or quasi-elastic light scattering and photon correlation spectroscopy, and is analogous to the Doppler shift of sound frequencies emitted from a moving source. Small particles or biomolecules diffuse quickly, causing rapid fluctuations of the scattered light. Larger particles, such as protein aggregates and nanoparticles, diffuse slowly, resulting in less-frequent fluctuations in the intensity of the scattered light.

These information-rich signals can be interpreted by means of a 1024-channel correlator and the PrecisionDeconvolve proprietary software that are components of the PDDLS/Batch system. Through use of a 100-mW laser with a fiber-optic-coupled high-speed photon-counting detector mounted at a 90° angle from the incident laser beam, the diffusion constant can be calculated as follows. The software statistically compares the intensity fluctuation-set measured over several microseconds with the next set and plots the decay over time. This autocorrelation function is then used to define the diffusion constant. From the diffusion constant, the particles' hydrodynamic radius is then calculated by means of the Stokes-Einstein equation.

Key advances of the DLS technology include the 1024-channel correlator, the technology's very high sensitivity, and its ability to operate in both a flow mode with an HPLC/SEC system and in a batch mode as described in this article. The cuvette-based batch system needs only 100 µl of sample to operate. A 25-µl cuvette is also available.

Batch R_h Distribution Assay by DLS

DLS detection can be applied to soluble and suspended particles with a hydrodynamic radius of 1.0 to 1000 nm. Typical smaller objects are therapeutic proteins that generally have an R_h in the range of 2.0 to 4.0 nm, and monoclonal antibodies (MAbs) at 4–8 nm. Assembling proteins, liposomal structures, and gold particles, by contrast, have R_h distributions ranging between 10 and 100 nm.

To determine the accuracy of the PDDLS/Batch molecular size analysis system, a polystyrene standard having a nominal particle size diameter of 90 nm (and thus a radius of 45 nm) was diluted as an aqueous suspension and introduced to the detector. The correlator was set for a sample time of 10 microseconds, a run time of 3 seconds, and a smoothing factor of 10. The last channel selected was channel number 1024. The assay time was only 1 minute. The sample distribution was monodisperse with a resultant R_h of 44.8 nm, as shown in Figure 2.

Figure 2. A 90-nm-diam polystyrene standard in aqueous suspension was measured by the DLS system. Run time was 1 minute. The resulting measured R_h of 44.8 nm correlates with the theoretical R_h of 45 nm.

The deconvolution feature of the DLS analyzer was also verified. A sample containing a distribution of particles in the 40- and 250-nm ranges was assayed. Figure 3 graphs the results of the 2-minute assay, showing the bimodal distribution clearly. The 40-nm particles were analyzed as having an R_h of 38.4 nm.

Figure 3. A bimodal polystyrene standard distribution was measured by the DLS system during a 2-minute assay. Particles with nominal hydrodynamic radii of 40 and 250 nm were resolved.

Also analyzed was a sample containing gold nanoparticles with E. coli–conjugated antibody (see Figure 4). This sample was retained from a batch that was sent to a diagnostic-kit-manufacturing facility prior to electron microscopy (EM) results being received. (The contract manufacturer did not have in-house EM capabilities.) The sample was diluted 1:100, with 100 µl used for the assay. The analysis time for the sample was 2 minutes. The PDDLS/Batch instrument calculated the hydrodynamic radius of the main particle distribution to be 32 nm, as expected. In addition, a distribution of smaller particles was found, clearly visible in the figure at around 2 nm. This contamination with smaller particles will cause a problem with this assay.

Figure 4. A 2-minute assay of E. coli MAb–conjugated gold particles reveals small-particle contamination at 2 nm.

In a similar analysis, graphed in Figure 5, a sample containing gold nanoparticles with Listeria-conjugated antibody was run. In this case, the size (R_h) of the gold nanoparticles was 52 nm. Again, the sample exhibited a small distribution of contaminants, this time at 9 nm.

Figure 5. A Listeria MAb–conjugated gold particle process sample contains a 9-nm contaminant.

Conclusion

An understanding of the relationship between the size of biomolecules and carrier particles in solution and product efficacy is a key factor in developing effective nanoparticle-based diagnostic tests. The use of DLS technology to determine the hydrodynamic radius and R_h distributions of nanoparticle formulations can provide rapid, accurate determinations of the stability of novel nanoparticle-based diagnostics for quality control purposes. The principal benefits of an instrument for low-nanometer-range particle sizing are its ease of use in comparison with traditional electron microscopy and its speed of analysis, which ranges from 1 to 5 minutes and is typically 2 minutes.

References

1. J Chandler, T Gurmin, and N Robinson, The Place of Gold in Rapid Tests, IVD Technology 6, no. 2 (2000): 37–49.

2. T Mourey and H Coll, "Size-Exclusion Chromatography with Two-Angle Laser Light Scattering of High-Molecular-Weight and Branched Polymers," Journal of Applied Polymer Science 56, (1995): 65.

3. JP Helfrich, "Flow-Mode Dynamic Laser Light-Scattering Technology for 21st Century Biomolecular Characterization," American Biotechnology Laboratory 16, no. 11 (1998): 64–66.

Illustration by James Schlesinger