Protein-modified ultra-small gold clusters for dual-modal in vivo fluorescence/photoacoustic imaging

Abstract

Background: Construction of nanoprobes for dual-modal fluorescence/photoacoustic imaging (PAI) is of great importance for the detection of disease pathology and the development of innovative therapeutics. Previously ultra-small gold clusters were designed and used as contrast agents for fluorescence imaging (FLI). However, it is not clear whether they can also serve as promising probes for PAI. In this study, protein-modified ultra-small gold clusters are produced and examined quantitatively as enhanced contrast agents for dual-modal in vivo fluorescence and PAI.

Methods: To construct the dual-modal ultra-small gold clusters, HAuCl₄·4H₂O aqueous solution was first mixed with the protein solution. NaOH was further introduced to the solution under vigorous stirring. The as-designed dual-modal nanoprobe was formed after stirring for 2 h at 65 °C. And then the solution was purified by gel column for further application. Zebrafish, cultivated in the solution containing gold clusters, was used in this study to demonstrate the dual-modal imaging ability of the nanoprobe by using our home-made optical-resolution photoacoustic microscopy and commercial fluorescence microscopy systems.

Results: The gold nanoclusters were synthesized with diameters of about 3 nm, which showed the broad absorption with a characteristic peak centered at 520 nm. A strong near-infrared emission ranging from 600 to 750 nm was also observed for the gold clusters. In addition, the cell viability was more than 90% even at a high concentration of the nanoprobes. The zebrafish cultivated with the gold clusters exhibited dramatically enhanced fluorescence and photoacoustic signal intensities.

Conclusions: Quantitative analysis results demonstrated that BSA-modified gold clusters were excellent contrast agents for in vivo dual-modal fluorescence/PAI. Due to their ultra-small size and superior biocompatibility, they can be applied to the detection and treatment of various diseases with enhanced sensitivity.

Keywords: Gold clusters; dual-modal nanoprobe; in vivo zebrafish imaging; optical-resolution photoacoustic imaging system.

Figure 1

The home-made optical-resolution PA microscopy system. NDF, neutral density filter; MS, motor stage; PH, pinhole; AMP, amplifier; FC, Fiber coupler; UT, ultrasonic transducer.

Figure 2

The fluorescence microscopy system.

Figure 3

Photograph of the representative zebrafish.

Figure 4

Characterization of BSA-Au clusters. (A) TEM image, (B) UV-Vis absorption and fluorescence emission spectra, (C) hydrodynamic diameter, and (D) zeta potential of the as-prepared BSA-modified gold clusters. Blue circles indicate the gold clusters.

Figure 5

Cell viability of 293T cells (A) and 4T1 cells (B) recorded after being incubated with BSA-modified gold clusters at various concentrations (0, 0.19, 0.38, 0.75, 1.5 mg·mL⁻¹).

Figure 6

In vivo FLI/PAI of zebrafish. In vivo FLI (top image), in vivo PAI of zebrafish (bottom image), and quantitative analysis of the fluorescence and PA signals (right). Noted that the white circles indicate the fluorescence or PA signals of the BSA-modified gold clusters. FLI, fluorescence imaging; PAI, photoacoustic imaging.