Synthesis of GNPs with NADH was accomplished by mixing small (100 μl) equal volumes of NADH and HAuCl4 solutions at room temperature (see Materials and Methods). When the NADH added was either equivalent or in excess of the molarity of the Au in solution, a pink to deep red color characteristic of plasmonic GNPs resulted within several seconds (Fig. 1A).
Spectra which corroborate these visual observations are presented in figure 1B, as the surface plasmon resonance (SPR) absorbance peak characteristic of GNPs (at approximately 530 nm) was found to increase in intensity with increasing concentration of NADH. This likely indicates that greater quantities of GNPs were produced as molar ratio increased. Supplementary figure A shows that this effect plateaued above a 4:1 molar ratio of NADH:Au3+, suggesting that complete reduction of available Au3+ occurred at this point. At molar ratios above 4:1 the location of the SPR peak did however, shift from its initial location, potentially indicating changes in the sizes or size distributions of the produced nanoparticles.
To further confirm the presence of GNPs in these solutions, several preparations were observed by transmission electron microscopy (TEM). In the micrograph displayed in figure 1C and throughout the data collected- it was found that all particles were spherical in shape- in contrast to other biosynthetic methods where there is high variability in shape. This relative regularity in shape may be due to the absence of confounding factors such as peptides, sugars, and salts that would inevitably be present in the crude extracts or cultures used in other methods. Energy-dispersive X-ray spectrometry of these same nanoparticles showed that they were indeed composed of Au (Fig. 1D).
Supplementary figure B shows that the size of GNPs as determined by TEM was found to be from 3 nm to 7.5 nm on average, but that average size was not found to correlate significantly with the molar of NADH:Au3+ (R2=0.4722). The lack of predictability in diameter observed is surprising, given that one would expect molar ratio to influence the relative rates of particle nucleation and growth, however others have also reported the absence of clear relationship between particle nucleation rates and final size. Particle size for the majority of GNP preparations was found to follow an approximately normal distribution at the 95% confidence level using the Anderson-Darling goodness of fit test. An example of such a distribution is shown in figure 1E (p=0.251 for normality). The presence of larger outlier particles in each preparation- for instance the 6.5–7 nm particles in supplementary figure B was also found to be typical among those imaged. The fact that all imaged preparations contained GNPs with small (<10 nm) average diameters agrees with the possibility that NADH is involved in other biosynthetic methods, as many of these also produce particles with average diameters under 15 nm.