The dimensions of the gold nanorod were chosen to be: length 80 nm, diameter 20 nm.

1. Synthesis of gold nanorods

Overview: The synthesis of gold nanorods encompassed two distinct steps: seed synthesis and seed growth. The seeds acted as a foundational platform for the subsequent growth of gold nanorods within the growth solution, ultimately leading to the obtainment of larger-sized gold nanorods.

1.1Preparation of seeds

The initial step in the synthesis of gold nanorods involved the preparation of gold seeds, serving as the initial nucleation points for the subsequent growth of gold nanorods. During the subsequent growth stages, gold atoms anisotropically adhered to the surface of these seeds, progressively growing until the desired dimensions of 80 nm in length and 20 nm in diameter for the gold nanorods were achieved. Consequently, the synthesis of gold seeds was pivotal to the successful fabrication of gold nanorods.

Jul. 1st, 2024

Three sets of gold nanorod were synthesized in parallel, and it was found that the color of this synthesized gold nanorod was too light and differed from the reasonable color compared with the colour showed in the literature, so the synthesis failed and the experimental operation needed to be improved. The potential failure may have been attributed to a lack of standardization in the preparation of the NaBH4 solution, which is inherently unstable and prone to deliquescence. Therefore, during the weighing process, it was imperative that NaBH4 be weighed only once and as swiftly as possible to minimize exposure. Additionally, prior to use, it should have been immersed in ice water to prevent deterioration.

Jul. 2nd, 2024

According to the new procedure, the color of the synthesized seed was dark brownish red, which was a relatively reasonable seed color. The seed synthesis can be considered successful and the growth of the gold-nanorods which is the following step of the experiment can be carried out.

1.2 Preparation of growth solution

Jul. 3rd, 2024

After the re-growth of the gold nanorods, the color of the solution was darker. Subsequently, 100 µL of this gold nanorod solution was withdrawn, diluted ten fold, transferred to a cuvette, and the UV-Vis-NIR absorption was monitored. The data obtained were organized and plotted as follows:

In the UV-Vis-NIR absorption spectrum of the product, the horizontal axis represented the incident wavelength (nm), while the vertical axis stands for the absorbance intensity. It was observed that two prominent absorption peaks were present at 522 nm and 760 nm. The peak at 522 nm indicated the presence of a significant quantity of spherical nanoparticles within the product mixture. Additionally, the absorption peak at 760 nm suggested that the aspect ratio (length-to-diameter ratio) of the gold nanorods did not match the targeted value of 4:1. Consequently, this synthesis was failed.In the synthesis process, AgNO3 served as a crucial agent for controlling the growth direction of the nanostructures. We hypothesized that the excessive production of gold nanorods indicated an error in the step involving the use of AgNO3. Given that AgNO3 solutions are prone to decomposition in the presence of light, they require airtight storage and protection from light. Prolonged storage further leads to a reduction in the effective concentration of AgNO3. Consequently, it was clear that the AgNO3 solution used in the initial experiment had been stored for an excessively long period. A freshly prepared AgNO3 solution is needed to solve this problem.

Jul. 6th, 2024

Given the inherent challenges in precisely controlling the synthesis size of gold nanorods, it was determined to be impractical to achieve gold nanorods with an optimal absorption peak at 800 nm, a length of 80 nm, and a diameter of 20 nm. Moreover, considering that variations in rod size had minimal impact on subsequent surface modifications, such as ligand exchange and sulfhydryl-DNA modification, we made a decision to go ahead with the gold nanorods we made to get familiar with the whole experiment.Furthermore, recognizing the limitation in synthesizing gold nanorods compared with the size we want, we decided to buy gold nanorods which have a more precise particle size and a more uniform particle size distribution for subsequent experiments which can further enhance the quality of the experimental outcomes.

Jul. 14th, 2024

The full band scan of gold nanorods is shown above.The absorption peak positions are located at 510 nm and 800 nm.

We tested the zeta potential of the purchased gold nanorods and the data are as follows:

2.Substitution of ligands on the surface of gold nanorods

Overview：Upon reviewing the literature, we concluded that gold nanorods with BSPP as the ligand could bind to sulfhydryl-DNA more easily. Therefore, following the synthesis of the gold nanorods, we designed the experiments to replace the ligand on their surface: from CTAB to BSPP. The structure of the ligand, BSPP, is showed in the Fig.9.

Jul. 20th, 2024

Based on the changes observed in the zeta potential before and after the process, it could be concluded that the ligand substitution was successful. However, it was noted that the concentration of the product obtained after concentration was not significant. It was hypothesized that during the procedure, a color change from orange to blue-violet occurred in the gold nanorods. Due to the subsequent increase in ionic strength within the solution, the stability of the gold nanorods was decreased, leading to the aggregation and denaturation. Consequently, a greater loss of gold nanorods was occured. Therefore, these steps should be avoided during the ligand exchange process.Between 0825 and 0915, we conducted three ligand substitution attempts, both before and after the main procedure, yet failed to achieve the desired results. Given that the ligand replacement step necessitated multiple centrifugations, sonication, and significant alterations in the solution's ionic strength, which could readily induce aggregation and precipitation of the gold nanorods, our team, following an extensive review of the literature, discovered that gold nanorods coated with sodium citrate ligands could be directly subjected to subsequent DNA modification steps without the need for ligand replacement. Consequently, we chosen to deleted the ligand replacement step.

3.Modification of sulfhydryl-DNA on the surface of gold nanorods

Jul. 30th, 2024

We found that after the modification steps, the absorption peak at 800 nm of the gold nanorod solution was significantly broadened and reduced in intensity, indicating a substantial concentration decrease and significant loss of gold nanorods during the process. We hypothesized that certain reagents utilized in the process were responsible for the aggregation and deposition of the gold nanorods. Consequently, further tests were conducted on all solutions involved in the process.

Aug. 4th, 2024

In our efforts to identify suitable buffers for resuspending the gold nanorods, we screened a variety of options. However, we discovered that several buffers caused the gold nanorods to aggregation, resulting in a change in color from reddish brown to blackish gray which were provided below:

From left to right, the gold nanorod solution was added to TCEP, TAE, NaCl, sulfhydryl-DNA, and TBE solutions. The sixth tube contained the gold nanorod solution alone for color comparison. After allowing the mixtures to stand at room temperature for 2 hours, it was observed that TAE and TBE solutions caused the color of the solution to change from light orange to blue-gray, indicative of gold nanorod aggregation and denaturation. Therefore, it was indicated that TAE and TBE buffers should be avoided as resuspension solutions.

Aug. 10th, 2024

The obtained data are shown below:

We conducted the modification of sulfhydryl-DNA onto the surface of gold nanorods and subsequently concentrated the product. After the concentration, an aliquot was withdrawn and diluted 20-fold for the purpose of assessing its absorption curve, as illustrated in Figure 16.Based on the data presented in Figure 16, the concentration of the product gold nanorods was calculated to be 1.04 nM (representing a 20-fold concentration of the tested sample). Notably, there was no changes in the absorption peak positions or peak shapes, indicating that the gold nanorods did not undergo aggregation and denaturation. Furthermore, the ratio of the primary to secondary peak heights ranged between 3 and 4, which is indicative of the high quality of the produced gold nanorods.

The hydration radius of the gold nanorods increased after the modification of sulfhydryl-DNA, and a change in the Zeta potential on their surface was observed, thereby providing evidence for the successful modification of sulfhydryl-DNA.

In summary of the aforementioned experiments, we successfully completed the synthesis of gold nanorods and the surface modification with sulfhydryl-DNA. Furthermore, we summarized and explored the standard experimental protocols, establishing a foundation for the subsequent binding of sulfhydryl-DNA modified gold nanorods to DNA-origami.