Preparation Procedure for Stable Gold Nanoparticle Conjugates

Topics Covered

Titration Procedure
Standard Gold Nanoparticles
Reactant-Free Gold Nanoparticles
Applications of Cytodiagnostics Gold Nanoparticles
Gold Nanoparticle Conjugate Preparation
Handling and Storage of Gold Nanoparticles
About Cytodiagnostics


Gold nanoparticle conjugates have found use in a variety of biological applications, including as probes in light and electron microscopy. Preparing stable gold nanoparticle conjugates is a simple process involving passive adsorption caused by electrostatic and hydrophobic interactions between the colloidal gold surface and the protein. This process is optimally achieved at a pH near the isoelectric point (pI) of the protein being conjugated.

The amount of protein adsorbed to the gold colloid is a critical parameter in the preparation of gold nanoparticle conjugates. If the amount of protein adsorbed to the gold surface is very small, aggregation takes place upon addition of electrolytes present in standard buffers. Hence, a titration is conducted to determine the amount of protein required for achieving protein concentration saturation and colloidal stability.

Titration Procedure

The titration process involves the following materials and instrument:

  • Standard Gold nanoparticles
  • Protein to be conjugated
  • Phosphate Buffered Saline, PBS
  • 10% PEG (5,000-20,000Da)
  • 10% NaCl
  • UV-VIS Spectrophotometer

Around 250μl of re-suspended gold nanoparticles is placed in 1.5ml Eppendorf tubes and is adjusted to a pH close to the pl of the protein to be conjugated. Then protein of varying amounts, from 0 to 1mg, is added into 25μl of gold nanoparticles during mixing to titrate the quantity required to saturate the gold surface. The mixture is incubated for 2-3 min at room temperature.

Then, around 250μl of10% NaCl solution is added and the color change is observed to determine the protein concentration at which the gold nanoparticle surface is saturated and aggregation is not taken place upon addition of 10% NaCl. This can be seen by an increase in absorbance at 580nm when compared to the control. It is to be noted that the protein concentration required for the saturation of the gold colloid can also be found by agarose gel electrophoresis caused by the change in charge upon the adsorption of the protein.

Standard Gold Nanoparticles

Cytodiagnostics makes its standard citrate stabilized spherical gold nanoparticles using a modified proprietary citrate reduction protocol. These nanoparticles are designed for high protein binding efficiency and are suitable for applications, including Surfaced Enhanced Raman Spectroscopy (SERS), dark field microscopy, lateral flow, and conjugate development.

With high shape uniformity, Cytodiagnostics colloidal gold nanoparticles reduce the inconsistency within an assay by facilitating control over the available surface area, while allowing protein absorption to the gold nanoparticles. This leads to higher performance for a specific assay at a lower cost. TEM images of gold nanoparticles are shown in Figure 1.

Figure 1. TEM images of gold nanoparticles

Cytodiagnostics supplies Standard Gold Nanoparticles in 0.1mM phosphate buffered saline (PBS) with a diameter range of 5-400nm. They are surface stabilized with citrate. Cytodiagnostics also offers these particles in a surfactant stabilized format.

Reactant-Free Gold Nanoparticles

Cytodiagnostics supplies its Reactant Free Gold Nanoparticles in 0.1mM phosphate buffered saline (PBS), with extensive purity of 99% free of residual reactants from production. The protein binding efficiency of these particles is at the same level of Cytodiagnostics Standard Gold Nanoparticles. They have been used in many different applications, including SERS, lateral flow, and conjugate development, with additional advantage of being reactant free for highly sensitive applications , including cell work and nanotoxicology analyses.

Applications of Cytodiagnostics Gold Nanoparticles

The following are the applications of Cytodiagnostics gold nanoparticles:

  • Nanotoxicology
  • SERS
  • Electron Microscopy
  • Biological Sensor Development
  • Lateral Flow Assays
  • Immunochromatography
  • Immunostaining
  • Conjugate Development
  • Cell Work

Gold Nanoparticle Conjugate Preparation

To prepare gold nanoparticle conjugate, the required amount of gold nanoparticles as decided by the specific application is transferred into a new tube. Then, 10% more protein is added along with the protein amount determined by the titration procedure. Then, the solution is continuously stirred while incubating at room temperature for 30min, followed by centrifuging at a speed appropriate to the size of the gold nanoparticles used for 30min. The pellent is then re-suspended in PBS supplemented with 0.1% BSA or 1% PEG. The resulting conjugate needs to be stored at 4°C until use.

Handling and Storage of Gold Nanoparticles

Gold nanoparticle products must be stored away from direct sunlight at 4-25°C as lower temperature increases product shelf life. Avoid freezing of gold nanoparticles as they will irreversibly aggregate if frozen. This can be observed by a change in color of the solution, as depicted in Figure 2. The colloidal gold can maintain its stability for at least one year when it is stored as specified.

Figure 2. Color Difference between Good Quality Gold Nanoparticles and Poor Quality Irreversibly Aggregated Gold Nanoparticles.

The gold nanoparticles may sediment at the flask bottom if stored for a prolonged period, especially larger gold nanoparticles. Hence, before use, the settled particles need to be re-suspended by swirling until obtaining a homogenous solution. Clean storage containers must be used for storing the colloidal gold to ensure optimal performance and stability.

For certain applications, it may be necessary to wash the gold nanoparticles before use. Centrifugation is the simplest method of removing potential contaminants in the nanoparticles solution. To achieve optimal performance, the centrifugation force needs to be adjusted as per the size of the gold nanoparticles used (Table 1). It is to be noted that re-suspension of non-functionalized gold nanoparticles must always be carried out in ultra-pure water to avoid irreversible aggregation owing to the fact of their sensitivity to salt containing buffers.

Table 1. Appropriate G Forces for Centrifugation of Gold Nanoparticles

Size (nm) Speed (g) Time (min)
5 100,000 30
10 17,000 60 (~50% recovery)
15 17,000 30
20 6,500 30
30 4,500 30
40 2,500 30
50 2,000 30
60 1,125 30
80 600 30
100 400 30
150 180 30
200 100 30

It is to be noted that recommended conditions are for 1ml volume and centrifugation by a microcentrifuge, except for 5nm gold nanoparticles that need an ultracentrifuge.

The centrifugation process involves placing aliquot of colloidal gold in suitable centrifuge tube, followed by centrifuging the gold nanoparticles at an appropriate G force depending the gold nanoparticle size for about 30min. The resulting supernatant is removed and re-suspended in appropriate volume of ultra-pure water, followed by re-dispersion of particles by vortex. The process is repeated as required.

About Cytodiagnostics

Cytodiagnostics is a biotechnology company based in Burlington, Ontario, Canada. We focus on providing and developing nanotechnology derived products and services for the international life science market. Our goal is to serve our customers with the highest quality products to ensure success in their research and development efforts.

This information has been sourced, reviewed and adapted from materials provided by Cytodiagnostics.

For more information on this source, please visit Cytodiagnostics.


  1. asmaa masoud asmaa masoud Egypt says:

    what is the best way to store nano gold and  the reason?

The opinions expressed here are the views of the writer and do not necessarily reflect the views and opinions of

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