Noninvasive and Spatiotemporal Control of DNAzyme-Based Imaging of Metal Ions In Vivo Using High-Intensity Focused Ultrasound

Abstract

Detecting metal ions in vivo with a high spatiotemporal resolution is critical to understanding the roles of the metal ions in both healthy and disease states. Although spatiotemporal controls of metal-ion sensors using light have been demonstrated, the lack of penetration depth in tissue and in vivo has limited their application. To overcome this limitation, we herein report the use of high-intensity focused ultrasound (HIFU) to remotely deliver on-demand, spatiotemporally resolved thermal energy to activate the DNAzyme sensors at the targeted region both in vitro and in vivo. A Zn²⁺-selective DNAzyme probe is inactivated by a protector strand to block the formation of catalytic enzyme structure, which can then be activated by an HIFU-induced increase in the local temperature. With this design, Zn²⁺-specific fluorescent resonance energy transfer (FRET) imaging has been demonstrated by the new DNAzyme-HIFU probes in both HeLa cells and mice. The current method can be applied to monitor many other metal ions for in vivo imaging and medical diagnosis using metal-specific DNAzymes that have either been obtained or can be selected using in vitro selection.

Figure 1.

Schematic of HIFU-activated noninvasive and spatiotemporal control of DNAzyme-based sensor for Zn²⁺ detection in vivo.

Figure 2.

Fluorescence response of the DNA probe and helper strand (a) before heat treatment, (b) after heat treatment at 43 °C for 30 min, and (c) when the heat-treated probe is added with zinc ions at 37 °C for 30 min.

Figure 3.

(a) Kinetics study of the stability of the probe before and after heat treatment at (a) 37 °C and (b) 43 °C. Kinetic study of the fluorescence signal change caused by the addition of zinc ions after heat treatment. (c) Zn²⁺-concentration-dependent response of probe after the thermal treatment. (d) FRET response of probe to different Zn²⁺ concentrations. (e) Calibration curve between F_A/F_D (=FI_666nm /FI_565nm) to Zn²⁺ concentration. (f) Fluorescence response of probe with HIFU activation. λ_ex = 514 nm.

Figure 4.

(a) Endogenous Zn²⁺ detection in HeLa cells. Scale bars: 20 μm. The fluorescence intensities from the CLSM images were quantified to calculate the (b) Cy3 signal and (c) FRET ratio (Cy5/Cy3) from (a). (d) Cellular images with additional exogenous 40 μM Zn²⁺ and (e) the corresponding intensity analysis of Cy3 signal (two-tailed Mann–Whitney t test, p < 0.001).

Figure 5.

HIFU-activated metal-ion sensing in vivo. Whole-body fluorescence imaging of mice after the injection of the probe and 40 μM Zn²⁺. (a) From the left flank and the right flank (probe and 40 μM Zn²⁺ injection), respectively. Whole-body fluorescence imaging of mice after the injection of the probe and Zn²⁺. (e) Quantification of fluorescence signal in mice before sonication (b), 25 min after sonication, (c) and 25 min after sonication and resting for 30 min (d) (p = 0.0223 calculated by an unpaired, two-tailed t test).