Interpreting Particle Spectra During Protein Conjugation
Colloidal stability is very important when optimizing a lateral flow assay. 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 unique optical signatures. 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 in the UV-vis spectra as a distinct red-shift in the UV-vis spectra. In the figures below the normalized UV-Vis spectra (each λ divided by λmax) of 80 nm gold and 150 nm gold nanoshells is 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 and a similar shift at 850 nm for the 150 nm gold nanoshells, 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 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). 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, or an elevated baseline or secondary peak will form at longer wavelengths (due to the formation of aggregates).
The plot above shows the UV-Vis spectra of a stable 80 nm gold conjugate, and an unstable 80 nm gold conjugate when the UV-Vis spectra is normalized (corrected by dividing the spectral values in each spectra by their respective λmax). Normalized spectra make it easier to observe any changes in the shape of peak shifts in the spectra. It is also useful to monitor UV-Vis spectra without normalizing. By only making adjustments 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.
A similar set of data is provided for 150 nm gold nanoshells where stabilized and destabilized spectra are compared both when the spectra are normalized and dilution corrected.
The normalized UV-vis spectra (corrected by dividing the entire spectra λmax) highlights the shape change and peak shift due to aggregation while the dilution corrected spectra highlights the drop in peak extinction.
The UV-Vis spectra is an indispensable tool for understanding the stability and conjugation state of reporter particles. By carefully interpreting the extinction spectra, there is a great deal of information that can be obtained. 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.