## 3 Results
### Characterization of Au Nanopeanuts
The resulting colloids prepared using 140 and 280 \(\mu\)L HAuCl\({}_{4}\) display stronger absorbance below 1000 nm, while a reverse appearance for wavelengths above 1000 nm shows greater amplitude with 420 and 700 \(\mu\)L additions (Figure S2b). Based on these observations in the change of gap and shell thickness, we speculate that the coupling of the central Au NR and Au/Ag nanoshell seems to strongly affect those two NIR bands. However, we should also be careful to consider the composition in Au/Ag nanoshells. The EDX analysis indicates that the ratios of Au/Ag shells were 11.9/88.1, 30.6/69.4, and 34.3/65.7 when 140, 280, and 420 \(\mu\)L of HAuCl\({}_{4}\) was used, respectively. To gain insight into the UV-Vis-NIR spectral tunability behavior, it is necessary to carry out theoretical calculations as well as more detailed experiments with systematic control of the gap relative to Au/Ag composition. For example, [PERSON] and coworkers developed a hybridization model to interpret plasmon modes for spherically concentric nanoparticles composed of Au nanoparticles surrounded by a silica layer and then an outer Au shell [24,25]. The plasmon resonance arises from the Au core to interact with the bonding and antibonding modes of the surrounding nanoshell. The final note for nanopeanut formation is that an additional feature was seen in the observation of the backfilling phenomenon from 420 to 700 \(\mu\)L. Figure S2 clearly shows the decrease in the gap and increase in the shell when subjected to 700 \(\mu\)L of HAuCl\({}_{4}\) addition. We found that both CTAB and AA in solution are essential to cause backfilling. With the current method of preparing nanopeanuts under a reaction solution containing HAuCl\({}_{4}\), CTAB, and AA, we could either fix the gap by changing the shell thickness or adjust the gap width by maintaining the shell thickness. The destruction of morphology normally occurs in the replacement reaction when a high HAuCl4 amount is used. To perform two-photon photosensization and two-photon luminescence imaging, the surface of the nanopeanuts, derived from the addition of 700 uL of HAuCl4, was conjugated with TBO and bis(amine)polyoxyethylene glycol (H2N-PEG-NH2, MW: 6000). After the centrifugation-wash process, as-prepared nanopeanuts were collected and redispersed in H2O for surface modification. First, 2-mercaptoethanol acid (MPA) was used to form an Au-S bond on the surface of nanopeanuts, resulting in the surface charge of nanopeanuts changing from 3.3 to \(-\)33 mV. Subsequently, the amine groups of TBO formed amide bonds with carboxylate groups of MPA through (1-ethyl-3-(3-dimethylaminopropyl)) carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) chemistry. The amount of immobi  
Figure 2: TEM images, colloidal aqueous solutions (inset), and UV-Vis-NIR absorption spectra of the resulting (**a**) Au NRs, (**b**) Au NR@Ag, (**c**) Au nanopeanuts, and (**d**) TBO-conjugated PEGylated Au nanopeanuts. lization was measured from the decrease in absorbance intensity at 630 nm of TBO left in supernatants and calculated based on a calibration curve according to TBO concentration (Figure S4). The -507 TBO was estimated to be on a single nanopeanut. The nanopeanut surface was not fully covered by TBO but rather had residual space left to tether hydrophilic amine-terminated PEG. Once again, amide bonds formed between MPA and PEG through the EDC/NHS reaction. The quantification of immobilized PEG was performed with a fluorescence-based method by the reaction of rhodamine 6G (R6G)-isothiocyanate with the exposed NH\({}_{2}\) group of PEG. The PEG conjugation amount was determined to be -2432 molecules per nanopeanut, which was derived from the difference in fluorescence intensity of R6G between the initial amount and final residue in supernatants, calculated based on a calibration curve according to the R6G concentration.