Introduction to Gold Nanoparticle Characterization

Background

Gold nanoparticles (AuNPs) exhibit broad application prospects in material science, biomedicine, and numerous other fields due to their unique physicochemical properties. The optical, electrical, and catalytic properties of AuNPs are highly correlated with their size (e.g., 10 nm vs. 100 nm) and morphology (spherical, rod-shaped, star-shaped, etc.) during processing, as well as their size (diameter), shape, surface structure, and aggregation state. When functionalized with surface-modified ligands (such as chemical groups or biomolecules), the presence of defects or oxide layers directly affects their stability, biocompatibility, and functionality. Therefore, it is necessary to characterize and evaluate the size and morphology of AuNPs, as well as the characterization results of surface functional modifications.

Ultraviolet-Visible (UV-Vis) Spectroscopy

Principle: When light irradiates the surface of AuNPs, free electrons in the nanoparticles generate collective oscillations under the electric field of the light, resonating with the frequency of the incident light. This phenomenon is known as surface plasmon resonance (SPR). The SPR effect of AuNPs causes them to produce a strong absorption peak at a specific wavelength, which serves as an important basis for characterizing AuNPs using UV-Vis spectroscopy.

Characteristics of Absorption Peaks: AuNPs with different sizes and shapes exhibit distinct positions and intensities of SPR absorption peaks. The wavelength of the peak absorbance increases with the increase in particle diameter. For particles with uneven shapes, such as gold nanobuds, the absorption peak position undergoes a red shift or multiple absorption peaks appear.

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Fig 1.The relationship between Jinnami particle size and surface plasmon resonance.

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Fig 2. The shape of gold nanoparticles depends the on local surface plasmon resonance, as shown by the visual appearance and UV visible spectra of spherical (A) and sea urchin-shaped (B) gold nanoparticles ("spiky gold").

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Fig 3. Normalized absorption spectra of 38 nm citrate stabilized AuNPs and PEG-AuNPs

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Figure 4. Visual appearance (left) and UV-Vis spectra (right) of monodisperse (A) and sodium chloride (NaCl) induced heavily agglomerated (B) 15nm gold nanoparticles.

Dynamic light scattering (DLS), as an important analytical technique, plays a key role in the research of gold nanoparticles (AuNPs).

Principle of Dynamic Light Scattering

DLS is based on the principle of Brownian motion of particles. When a laser beam irradiates particles in a dispersed system, the particles continuously change their positions due to Brownian motion, causing the phase and intensity of the scattered light to fluctuate over time. By measuring the fluctuations in the intensity of these scattered lights over time and analyzing them using correlation functions, the diffusion coefficient of the particles can be obtained. According to the Stokes-Einstein equation, the diffusion coefficient is related to the hydrodynamic radius of the particles, allowing the calculation of the particle size distribution.

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Figure 5. Scattering Image Size

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Figure 6.histogram obtained by dynamic light scattering measurement of 20 nm gold nanoparticles before (blue) and after (green) surface functionalization with a 3kDa PEG-thiol. The hydrodynamic size increased from 30 nm to 48 nm through the addition of a PEG-layer.

Microscopic Imaging of Gold Nanoparticles

a.Dark-Field Microscopy Imaging

Dark-field microscopy employs a special optical path design that allows only scattered light to enter the objective lens for imaging while blocking background light. Due to their light-scattering properties, gold nanoparticles (AuNPs) generate bright signals under dark-field microscopy, forming a sharp contrast with the dark background. AuNPs exhibit surface plasmon resonance (SPR) characteristics: when light irradiates the nanoparticles, free electrons on their surface undergo collective oscillations, resonating with the frequency of the incident light, leading to strong absorption and scattering of light at specific wavelengths. This property enables AuNPs to serve as excellent contrast enhancers or signal sources in microscopic imaging, thus facilitating their application in dark-field microscopy.

b.SEM and TEM

TEM (Transmission Electron Microscopy)

TEM enables high-resolution characterization of AuNP morphology, size, and distribution. For instance, it directly observes their morphology during composite material synthesis. High-resolution TEM further analyzes internal structures such as crystal lattices, aiding in studies of growth mechanisms.

SEM (Scanning Electron Microscopy)

SEM visualizes surface topographical features (e.g., roughness) and investigates AuNP distribution and aggregation states on substrates, thereby optimizing properties in applications like catalysts.

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Figure 7. TEM image

Gel Electrophoresis

As a common separation technology, gel electrophoresis plays an important role in the research of gold nanoparticles, which can be used to separate gold nanoparticles with different shapes and sizes, and analyze their interactions with biomolecules.

The following two types of gel electrophoresis are mainly used:

Type	Principle	Applicable scenarios
Agarose gel electrophoresis	Using agarose to form a porous network for separation based on particle size differences, with larger pore sizes suitable for separating larger sized (>100 nm) gold nanoparticles or aggregates.	Preliminary screening for aggregation status and separation of large-sized particles.
Polyacrylamide gel electrophoresis (PAGE)	The pore diameter of polyacrylamide gel is more uniform and adjustable, and the gold nanoparticles of nanometer size (10-100 nm) can be separated by controlling the crosslinking degree, with higher resolution100 nm��	Accurately analyze the size of monodisperse particles and the thickness of the surface modification layer.

Bio-Functionality Testing of Gold Conjugates

Due to the unique properties of gold nanoparticles and their coupling with other biomolecules, gold conjugates have been widely used as probes in various biological assays and have shown promising applications in the biomedical field.

The biological functional testing of gold conjugates covers cellular, molecular, in vivo, and other levels, as follows:

1.Cell level testing

Cell uptake test

Cytotoxicity testing:

Cell function impact test

2. Molecular level testing

Biomolecular binding specificity testing

Biomolecular activity testing

3. In vivo testing

Animal model construction

In vivo distribution and targeting testing

Therapeutic effect testing