Interpreting Particle Spectra During Protein Conjugation

It is essential that particle conjugates are stable before they are incorporated into a lateral flow assay as unstable particles will aggregate on the test strip and reduce assay performance. A simple method for evaluating successful conjugation and conjugate stability is to measure and compare the ultraviolet-visible (UV-Vis) spectra before and after conjugation.

Gold nanoparticles absorb and scatter light with extraordinary efficiency and have a color that is a function of their size and shape. Their strong interaction with light occurs because the conduction electrons on the metal surface undergo a collective oscillation when they are excited by light at specific wavelengths. This oscillation is known as a surface plasmon resonance (SPR). The SPR is sensitive to changes in the particle aggregation state and the local refractive index near the particle surface. During conjugation the particle spectra can be monitored to determine if the particles are colloidally stable in solution and if antibody has successfully bound to the particle surface.

The Effects of Conjugation on the Optical Properties of Gold Nanoparticle Reporter Particles

After a successful conjugation, there is a change in the local refractive index which can be observed as a distinct red-shift in the UV-vis spectra (i.e. the peak wavelength increases). In the figures below, the normalized UV-Vis spectra (each OD value divided by the OD at λmax) of 80 nm gold spheres and 150 nm gold nanoshells are shown before and after conjugation. Notice that there is a 2-3 nm red shift at the peak in the spectra, around 550 nm for the 80 nm gold (figure 1) and 850 nm for the 150 nm gold nanoshells (figure 2), but the overall shape of the spectra remains the same before and after conjugation. Additionally, another peak appears at 280 nm, which is due to absorbance of free protein in the conjugate diluent/storage buffer.

The Effect of Flocculation and Aggregation on the Optical Properties of Reporter Particles:

When gold nanoparticle solutions are destabilized, they can flocculate (reversibly bind together) or aggregate (irreversibly bind together). This may occur during conjugation when parameters and conditions, such as reaction buffer or antibody loading, are suboptimal.

Visible signs of aggregation include excessive plating on the tube following centrifugation, noticeable color change of the particle solution, a drop in the optical density of the solution and formation of particulates. The optical properties of gold nanoparticles change when particles associate due to a modification of the surface plasmon resonance where conduction electrons near each particle surface become delocalized and are shared amongst neighboring particles. When this occurs, the surface plasmon resonance shifts to lower energies, causing the absorption and scattering peaks to red-shift to longer wavelengths.

UV-Visible spectroscopy can be used as a simple and reliable method for monitoring the stability of nanoparticle solutions. As the particles destabilize, the optical density (OD) will decrease due to the depletion of stable nanoparticles, and often the peak will broaden. An elevated baseline or secondary peak may also form at longer wavelengths due to the formation of aggregates.

The plot above shows the normalized UV-Vis spectra of a stable 80 nm gold conjugate, and an unstable 80 nm gold conjugate. While there is only a slight change in the peak location (small red shift), there are other important changes to the spectra. The full-width-half-max of the peak is increased (the peak broadens), there is a change in the “peak-to-trough” ratio which for gold nanoparticles we define as ratio between the peak extinction and 480 nm, and finally, there is an increase in the long wavelength “tail” out through 1000 nm.

Normalized spectra make it easier to observe any changes in the shape of peak shifts in the spectra, but it is also useful to monitor UV-Vis spectra without normalizing. By only adjusting for changes in dilution, the UV-Vis spectra will highlight changes to the absorbance (optical density) of the reporter particle which are particularly apparent when the particles have aggregated.

Here, the peak extinction value at 550 nm is indicative of aggregation but it is important to look at the shape of the entire spectra instead of just a peak drop. In some cases, (such as plating on the tube), the peak absorbance will drop but the spectral shape will remain constant since the particle in solution remain unaggregated. It is important to differentiate between different modes of peak extinction drop in order to determine how to mitigate the effect.

While the examples shown above are a relatively extreme case where the changes in the spectra are dramatic, smaller changes in the UV-Visible spectra can be used to make a determination on whether one set of conjugates is better than another.

For example, measuring the ratio between 550 nm and 650 nm, the magnitude of the extinction at 800 nm, or the “peak-to-trough” ratio between 550 nm and 450 nm are numerical values that can be used to look for subtle changes in the aggregation state of the particles.

The table below (figure x) shows these ratios for the stable and unstable conjugates. For 40 nm and 80 nm gold we prefer monitoring the Ratio of OD at 550/700 nm and the OD of the Tail as the primary metrics for determining whether one conjugate is better than another by UV-Vis.

A similar set of data is provided for 150 nm gold nanoshells above where stabilized and destabilized spectra are compared both normalized and dilution corrected. When aggregated, the nanoshell spectral peak will broaden and be reduced in intensity. The peak to trough ratio for nanoshells is defined to be the ratio of the highest extinction value (around 800 nm) and the smallest extinction value at lower wavelengths (around 510 nm). High quality nanoshells will have peak to trough ratios above 3 with the best nanoshells having peak to trough ratios in the 4 to 5.7 range. Monitoring the peak to trough ratio is our preferred method of determining whether a nanoshell solution has become destabilized. In the example above, the peak OD at 800 nm and the Peak-To-Trough ratio are provided below.

The UV-Vis spectrum is an indispensable tool for understanding the stability and conjugation state of reporter particles. By carefully interpreting the extinction spectra, a great deal of information can be obtained that will be useful early on in conjugate optimization, as a stable conjugate is essential to get high quality, consistent results on a lateral flow assay.

In some cases, UV-Vis data is just as useful as much more expensive nanoparticle characterization techniques such as Dynamic Light Scattering, Zeta Potential, Transmission electron microscopy, etc. We strongly encourage anyone who is working with nanoparticle probes to purchase and use a spectrometer that can measure the full spectral response of their conjugates.