Gold nanoparticles have many applications in biology and medicine. Gold nanoparticles are used as biosensors, cellular probes, drug delivery vehicles, or as optical contrast agents (1). To use gold nanoparticles for these specific applications, the ability to modify the surface is extremely important. A modification of the surface with polymers such as polyethylene glycol (PEG) and polyethylene oxides reduce non-specific binding, small molecules such as biotin and peptides and proteins can provide them specificity toward cellular targets in vitro and in vivo, and DNA enables them to be used for genetic detection. A key strategy to modifying the surface of the gold nanoparticles is with molecules that contain sulfur, nitrogen, or oxygen atoms as they have high binding affinity to gold atoms. As a result, thiolated-DNA and proteins adsorb onto the surface of the gold nanoparticles relatively easily. The design of Cytodiagnostics gold nanoparticles allows for the use of either adsorption to coat polymers or bio-recognition molecules on the surface, by covalent conjugation, or by using biological interactions (e.g., streptavidin-biotin). This provides greater versatility in the design of you gold nanoparticle system. This technical note provides a brief description of the current modification strategies and some of the emerging biomedical applications of these materials.
PEG is a polyether molecule and is typically described by the molecular weight and whether they are linear, branched, star, or combed-shaped. PEG molecules can also be functionalized with thiols, amines, carboxylic acids, or alcohols. PEG molecules are coated onto gold nanoparticles by a sulfur-gold atom bond. Interestingly, coating of a dense layer of PEG onto gold nanoparticles has shown to reduce non-specific binding of proteins (2). It was recently shown that gold nanoparticles with greater than 0.96 PEG/nm2 is required to reduce non-specific binding and to inhibit their uptake into macrophage cells. Using these results, we designed PEGylated gold nanoparticles with the lowest non-specific cellular uptake. This is very important when using gold nanoparticles in biology where the particles are programmed to target specific molecules or cellular receptors. Non-specific protein binding can affect the specificity. In addition, our gold nanoparticle-PEG system contains protruding carboxylic acids or amines that allows other molecules to be conjugated to the surface by using the coupling agent 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide, better known as EDC. In this reaction, the gold nanoparticles is incubated with EDC and the biological molecule of interest for 2 hrs and then purified by centrifugation to remove excess biological molecules (see Tech Note 105 - Covalent conjugation to Cytodiagnostics Carboxylated Gold Nanoparticles for detailed procedure). These gold nanoparticle conjugates are then ready to be used for biological purposes.
Gold nanoparticles cannot recognize specific biological targets without surface modification. It is well known that single-stranded DNA’s called oligonucleotides can recognize a complementary sequence, antibodies recognize antigens, and peptides recognize antibodies. By coating the surface of gold nanoparticles with oligonucleotides, antibodies, peptides, or other bio-recognition molecules, one is then able to recognize specific targets in solution, in western blots (see figure 1), on/in cells, or in tissues in animals. Basically, these molecules provide gold nanoparticle with a biological function. Typically these bio-recognition molecules are coated onto the surface via direct adsorption or by covalent conjugation to the gold nanoparticle surface containing carboxylic acid or amine functional groups.
Figure 1. Immuno-dot blot assay for detection of human IgG antibodies using gold nanoparticles, silver nanoparticles and gold nanourchins ("spiky gold") coated with bio-recognition molecules. Note how the difference in appearance (color) of the dots can be achieved using different types of noble metal protein conjugates varying in either shape or composition.
Engineering a gold nanoparticle surface that contains PEG and bio-recognition molecules is ideal for their application. The PEG molecule prevents non-specific binding while the bio-recognition molecule provides biological specificity. Cytodiagnostics gold nanoparticle products enables researchers to design their nanoparticles in this manner, for optimum biological use.
Gold nanoparticles coated with PEG or/and bio-recognition molecules (BRM) has many applications, e.g. biosensors, cellular probes, drug delivery vehicles, or as optical contrast agents. Below, we provide some specific examples of how to use gold nanoparticle conjugates.
Single stranded DNA-coated onto gold nanoparticles can be used for detection of genetic material (3). In this application, single stranded oligonucleotide-coated gold nanoparticles are incubated with a DNA fragment of interest. If the fragment is complementary to the oligonucleotide sequence on the gold nanoparticles, particles are assembled together. As a result of this "aggregation” the color of the solution changes from red to blue because the surface plasmon is coupled when particles are in the aggregated state. The color change thus indicates a positive detection. Mutations can be detected by heating the sample. DNA de-hybridizes when heated and when a mutation is present in the sequence, the melting temperature is lowered. By measuring and comparing the temperature of a mutated sequence to a perfectly complementary sequence, one is able to detect whether a mutation is present or not. More complex schemes can also be designed to identify the location of a mutation within the DNA fragment analyzed.
Through years of experience, Cytodiagnostics has optimized protocols for conjugation of oligos to gold nanoparticles and we are proud to offer our OligoREADY™ conjugation kits for easy conjugation of thiolated oligonucleotides to gold nanoparticles with sizes in the range of 5-100nm.
The gold nanoparticle PEG/BRM system can be designed to selectively target tumors and bind to cancerous cells. Interestingly, PEGylated gold nanoparticles can target tumors by a passive mechanism alone. An advantage of the protective PEG-layer on the gold nanoparticles is that it has been shown to reduce macrophage uptake. Macrophages are part of the reticuloendothelial system that removes foreign materials from the blood. The PEGylated layer reduces the interaction of the gold surface with blood proteins thereby minimizing non-specific macrophage uptake. This allows the nanoparticles to reside in the blood for long-term, which allows for a greater chance of tumor extravasation. If coated with a drug or imaging agent (see our Fluorescent Gold Nanoparticles), the gold nanoparticles can be used as a visualization tool as well as a delivery vehicle to the tumor. Another method of targeting tumors is by coating the gold nanoparticles with a bio-recognition molecule that recognizes receptors on tumor cells, the extracellular matrix, or blood vessels, i.e. active targeting. The advantage of using gold nanoparticles is that one can control the delivery efficiency by the size, shape, or surface chemistry (4).
One of the key questions facing researchers is to understand how the size, shape, and surface chemistry (known as the physico-chemical properties) affect how nanoparticles distribute in the cell and body, and whether specific nanomaterial designs cause toxicity. Gold nanoparticles are ideal platforms to perform these studies on because they can be synthesized and tuned with a narrow size distribution. Further, designs with different shapes and surface chemistries can be readily achieved. This allows one to systematically evaluate their behavior in biological systems. By performing such studies, one is then able to identify the designs with the lowest toxicity, which subsequently can be selected for the final application(s).
Gold nanoparticles scatter light, and when using a dark field microscope to image them, they appear as bright spots (similar to fluorescence). A unique advantage is that the dark field signal does not photobleach like fluorescence from dyes. By labeling gold nanoparticles with bio-recognition molecules such as an antibody to a cell surface receptor etc. cells can thus conveniently be targeted and imaged. In addition, low-level target detection can be improved by using silver enhancement of bound gold nanoparticles.
The surface is the interface of the nanoparticle with the environment and the ability to manipulate the surface of gold nanoparticles is essential for their use. Here, we present a product with a PEGylated surface that reduces non-specific binding to the gold surface and further functionalization with bio-recognition molecules provides an easy strategy to both target and visualize cellular targets and intracellular processes.