The fluorescence of a fluorophore can be artificially altered by influencing excitation/emission states of its electrons, such as placing it in an electromagnetic (EM) field. This phenomenon can be beneficially used in developing an efficacious fluorescent contrast agent for biosensing and molecular imaging. Good candidates for providing EM field in a local level are nano metal structures generating strong EM (plasmon) field upon receipt of light. Gold nanoparticle (GNP) is ideal for the purpose because of its inert and non-toxic nature, and its surface properties accommodating easy conjugation of biomolecules.
The three main groups of the parameters affecting fluorescence emission level by GNP are: (1) GNP properties: the size of the GNP (core-to-shell ratio if the particle is hollow) and the wavelength of the incident light [in a practical sense, it is the excitation (Ex) light wavelength of the fluorophore]; (2) Fluorophore properties: the wavelength of the emission peak (Em) and the quantum yield; and (3) The distance between a fluorophore and a GNP. We have theoretically analyzed the fluorescence change by GNP, with respect to these parameters, for the fluorophores that are frequently used in biomedical sensing/imaging. For the fluorophores with Ex/Em wavelengths near the plasmon resonance peak of the GNP (~520 nm) [e.g., FITC, AF532, Cy3, etc.], the fluorescence is extensively quenched when the fluorophore is placed within 10 nm from GNP. This means that, for these fluorophores, GNP can be used as an effective quencher. For those with the Ex/Em wavelengths away from the resonance peak [e.g., AF647, AF700, ICG, etc.] the fluorescence is quenched when the fluorophore is very close (less than a few nanometers) to a GNP. At a particular distance between the fluorophore and a GNP, the fluorescence can be significantly enhanced, and the level of the enhancement can be strongly dependent on the GNP size. For bigger GNPs, the distances for both quenching and the maximum enhancement are shorter, and the enhancement level is greater.
With the knowledge obtained from the theoretical analysis, we have designed optical contrast agents of Cypate, an NIR fluorophore, with spherical solid GNPs and hollow gold nanospheres (HGNs). Cypate is excellent for in vivo application because it is a derivative from FDA approved flurophore, ICG. Most natural fluorescence generated by tissues is in UV or visible wavelength and, using a NIR fluorophore can avoid the tissue generated fluorescence. Also NIR can penetrate into the tissue deeper than visible light. Cypate was conjugated to GNPs/ HGNs of various sizes via spacers at various lengths, and its fluorescence level was observed. Utilizing the GNP’s fluorescence quenching effect when fluorophore is very close to GNPs, an enzyme-triggered contrast agent was developed by placing Cypate to GNP/HGNs via a very short and substrate containing spacer. The enzyme used for our system was urokinase type tissue plasminogen activator (uPA), an enzyme secreted by highly invasive breast cancer. This agent emitted little fluorescence. In the presence of uPA, the spacer was cleaved and the fluorescence was restored. In addition, to utilize the GNP’s fluorescence-enhancement property, Cypate was conjugated to GNPs/HGNs via spacers at particular lengths and we were able to enhance Cypate emission level extensively.
Fluorescence tailoring using GNPs can be realized by appropriately selecting fluorophores and manipulating the parameters associated. This mechanism can be used in developing optical contrast agents that are conditionally quenching or emitting, and that are with enhanced sensitivity.
The authors acknowledge the financial support from U.S. Army Breast Cancer Program (BC074387).