Seed‑Mediated Synthesis of Tunable‑Aspect‑Ratio Gold Nanorods for Enhanced Near‑Infrared Photoacoustic Imaging

Background

One‑dimensional nanostructures—nanowires, nanorods, nanotubes, and nanobelts—offer anisotropic geometries that are ideal for nanodevices and biomedical applications. Gold nanorods (AuNRs) are especially attractive because their shape‑dependent surface‑plasmon resonance (SPR), facile synthesis, and amenability to surface modification make them ideal contrast agents for PAI and photothermal therapy. Recent advances have enabled precise tuning of the longitudinal SPR by controlling the aspect ratio, enabling resonance within the optical windows of biological tissue (700–950 nm and 1000–1350 nm). However, scalable synthesis of high‑aspect‑ratio AuNRs with broad NIR absorption remains a challenge.\n

In this work, we report a straightforward seed‑mediated method that yields AuNRs with aspect ratios from 8.5 to 15.6 by modulating ascorbic acid concentration. The resulting rods display a tunable NIR absorption band spanning 680–1100 nm and, when PEG‑functionalised, demonstrate negligible cytotoxicity and robust in vivo imaging performance.\n

Experimental

Synthesis of Gold Nanorods

The modified seed‑mediated protocol follows established procedures (Refs. 16,17). Briefly, 10.3 mL of 0.025 M HAuCl₄ and 3.644 g CTAB were mixed, diluted with deionised water to 2.5×10⁻³ M HAuCl₄ and 0.1 M CTAB, and aliquoted into flasks A–D. A 350 µL ice‑cold 0.01 M NaBH₄ seed solution was added to flask A and stirred for 3 min. Successive aliquots (0.4 mL) of flasks A, B, and C were transferred to B, C, and D, each receiving 25 µL of 0.1 M ascorbic acid (AA). After a 5‑s stir, 4 mL from flask C and 250 µL AA were added to flask D, which was then left at 28 °C for 12 h. The resulting precipitate was washed repeatedly with distilled water to remove excess CTAB, yielding the typical nanorods (AuTR).\n

By varying the AA volume—35 µL, 30 µL, 20 µL, and 15 µL in the four‑step sequence—aspect ratios of 8.5, 10.5, 12.5, and 15.6 were achieved, respectively.\n

Surface Modification of AuNRs

SH‑PEG (10 mg in 1 mL water) was sonicated for 10 min and then reduced with 50 mL 0.1 M NaBH₄ under sonication for 15 min to break disulfide dimers. Cleaned AuNRs were mixed with the SH‑PEG solution (10 mL) and stirred for 5 min, then left for 5 h. The final product was purified by centrifugation and washing with water.\n

Characterization Methods

Morphology was examined by SEM (JEOL JSM‑7001F) and TEM (JEOL 2100F, 200 kV). UV‑vis spectra were recorded with a Shimadzu 3100 spectrophotometer. Photoacoustic signals were measured using a rotation‑scanning PA system equipped with a Surelite I‑20 laser, Surelite OPO Plus, Olympus PMUT V310‑SU transducer, and data‑acquisition hardware.\n

Cell Viability Experiments

All procedures received IACUC approval at Taiyuan University of Technology. HeLa cells were incubated with AuNRs (0.25–1.0 nM) for 24 h, and viability was assessed via the MTT assay and optical microscopy.\n

In Vitro and In Vivo PAI

Agar phantoms (2 g agar in 100 mL water) were prepared by heating, stirring, and casting into a 4.5 cm mold. A 0.9‑mm glass capillary filled with fresh ox blood or blood mixed with AuTR‑PEG (1 nM) was inserted to mimic a vessel. The phantom was illuminated with 680‑nm or 800‑nm lasers (11 mJ cm⁻²). For animal imaging, mice were anesthetised with isoflurane and chloral hydrate, hair was removed, and an ultrasonic coupling agent was applied. After intravenous injection of 1 nM AuTR‑PEG, cerebral vessels were imaged at 800 nm and 680 nm. A second injection was performed 24 h later for the alternate wavelength.\n

Results and Discussions

High‑resolution TEM confirmed uniform AuTRs with diameters of 22 ± 1.5 nm and lengths of 290 ± 13 nm (aspect ratio ≈13.2). HRTEM revealed (110) lattice fringes (1.44 Å), indicating growth along the [110] direction.\n

UV‑vis spectra displayed two peaks: a transverse peak near 520 nm and a longitudinal peak that red‑shifted from ~680 nm to 1100 nm as the aspect ratio increased from 8.5 to 15.6, matching the biological NIR window. SH‑PEG functionalisation caused a minor (~5 %) reduction in peak intensity without shifting the position.\n

In vitro PA experiments showed linear signal dependence on AuTR concentration. At 800‑nm excitation, AuTRs produced a 2.3‑fold PA signal increase versus blood alone; Au rod 1 (aspect ratio 8.5) yielded a 2.1‑fold increase at 680 nm. Multi‑wavelength PA spectra matched the optical absorption, confirming the correlation between SPR and PA performance.\n

MTT assays revealed that AuTR‑PEG maintained >95 % cell viability at 1 nM, whereas unmodified AuTR reduced viability to 71 % at the same concentration, indicating the protective effect of PEGylation.\n

In vivo imaging demonstrated that AuTR‑PEG (800 nm) and Au rod 1‑PEG (680 nm) significantly enhanced vessel contrast: the AuTR‑PEG/800 nm group achieved a 3.1‑fold contrast increase and a 5.6‑fold SNR boost relative to controls. The Au rod 1‑PEG/680 nm group achieved 3.6‑fold contrast and 5.7‑fold SNR improvement.\n

Typical morphology and structure of Au nanorod synthesized at 25 µL 0.1 M AA (AuTR): a Bright‑field TEM image. b Amplification TEM image, of a single rod. c HRTEM image of a single rod in rectangular area “1” from panel b. d UV‑vis absorption spectra of AuTR and AuTR‑PEG

Conclusions

We have developed a scalable, seed‑mediated synthesis that produces gold nanorods with aspect ratios ranging from 8.5 to 15.6, yielding tunable longitudinal SPR from 680 to 1100 nm. SH‑PEG functionalisation confers excellent biocompatibility and stability, enabling robust NIR photoacoustic contrast. In vitro and in vivo results confirm superior contrast and SNR in PAI, validating these nanorods as versatile agents for imaging across the first NIR window, with potential applications in cerebrovascular disease diagnostics.\n

Abbreviations

1D

One‑dimensional

AA

l(+)-ascorbic acid

AuNR

Au nanorod

AuTR

Au typical nanorods

CTAB

Cetyl trimethylammonium bromide

DNA

Deoxyribonucleic acid

MTT

Methyl thiazolyl tetrazolium

NIR

Near‑infrared

NRs

Nanorods

OPO

Optical parametric oscillator

PA

Photoacoustic

PAI

Photoacoustic imaging

SAED

Selected area electron diffraction

SEM

Scanning electron microscopy

SH-PEG

Thiol‑polyethylene glycol

SNR

Signal‑to‑noise ratio

SPR

Surface plasmon resonance

TEM

Transmission electron microscopy