Synthesis and bioconjugation of gold nanoparticles as potential molecular probes for light-based imaging techniques

Abstract

We have synthesized and characterized gold nanoparticles (spheres and rods) with optical extinction bands within the "optical imaging window." The intense plasmon resonant driven absorption and scattering peaks of these nanoparticles make them suitable as contrast agents for optical imaging techniques. Further, we have conjugated these gold nanoparticles to a mouse monoclonal antibody specific to HER2 overexpressing SKBR3 breast carcinoma cells. The bioconjugation protocol uses noncovalent modes of binding based on a combination of electrostatic and hydrophobic interactions of the antibody and the gold surface. We discuss various aspects of the synthesis and bioconjugation protocols and the characterization results of the functionalized nanoparticles. Some proposed applications of these potential molecular probes in the field of biomedical imaging are also discussed.

Figure 1

Optical extinction spectrum of preformed 8-minute-aged CTAB-capped gold nanospheres as seed for nanorod synthesis.

Figure 2

Gold nanorods synthesized using 50 μL of silver nitrate in growth solution. (a) Optical extinction spectrum showing the transverse plasmon peak at 516.5 nm and the longitudinal plasmon peak at 675 nm. The amplitude of the longitudinal plasmon peak is higher than transverse plasmon peak which indicates the formation of high yield of nanorods compared to spheres. (b) High-resolution scanning electron microscope (SEM) image of gold nanorods showing high monodispersity. Few nanospheres are observed.

Figure 3

Gold nanorods synthesized using 250 μL of silver nitrate in growth solution. (a) Optical extinction spectrum showing the transverse plasmon peak at 516 nm and the longitudinal plasmon peak at 850 nm. (b) High-resolution scanning electron microscope (SEM) image.

Figure 4

Histograms of gold nanorod aspect ratios synthesized with (a) 50 μL silver nitrate, mean aspect ratio of 2.3 ± 0.3 (mean length 44.8 ± 4.1nm, mean width 19.8 ± 2.9 nm); and with (b) 250 μL silver nitrate, mean aspect ratio of 3.6 ± 0.6 (mean length 51.0± 4.4 nm, mean width 14.1±2.1).

Figure 5

Normalized extinction spectra of gold nanorods with increasingly red-shifted longitudinal plasmon bands, synthesized using 50, 100, 150, 200, and 250 μL of silver nitrate in the growth solution for curves 1–5, respectively. Normalization of the spectra is done with respect to the transverse plasmon peak amplitudes.

Figure 6

Extinction spectra before and after incubation of HER81 with (a) gold nanospheres, (b) gold nanorods. In both cases, a red shift in plasmon band(s) occurs after incubation with the antibody signifying successful bioconjugation.

Figure 7

Confocal reflectance images (left) and bright field images (right) of (a) SKBR3 cells incubated with silver-stained HER81/gold sphere conjugates, (b) CHO cells under the same conditions. Care was taken to maintain the same acquisition parameters in both cases. The silver-stained bioconjugates are detected at the cell membranes of SKBR3 cells where HER2 is localized. This indicates successful conjugation and retention of functionality of the antibody after conjugation. No such accumulation of HER81/gold sphere conjugates is demonstrated in HER2 negative CHO cells.

Figure 8

Corresponding images as in Figure 7 for bioconjugates consisting of silver-stained HER81/gold nanorod conjugates incubated with (a) SKBR3 cells and (b) CHO cells. Care was taken to maintain the same acquisition parameters in both cases. The bioconjugates are accumulated at the cell membranes of SKBR3 cells where HER2 is localized. As expected, no such accumulation takes place in the case of HER2 negative CHO cells.