Quantitative detection of microRNA-21 in vivo using in situ assembled photoacoustic and SERS nanoprobes

Abstract

Accurately quantifying microRNA levels in vivo is of great importance for cancer staging and prognosis. However, the low abundance of microRNAs and interference from the complex tumor microenvironment usually limit the real-time quantification of microRNAs in vivo. Herein, for the first time, we develop an ultrasensitive microRNA (miR)-21 activated ratiometric nanoprobe for quantification of the miR-21 concentration in vivo without signal amplification as well as dynamic tracking of its distribution. The core-satellite nanoprobe by miR-21 triggered in situ self-assembly was built on nanogapped gold nanoparticles (AuNNP probe) and gold nanoparticles (AuNP probe). The AuNP probe generated a photoacoustic (PA) signal and ratiometric SERS signal with the variation of miR-21, whereas the AuNNP probe served as an internal standard, enabling ratiometric SERS imaging of miR-21. The absolute concentration of miR-21 in MCF-7 tumor-bearing mice was quantified to be 83.8 ± 24.6 pM via PA and ratiometric SERS imaging. Our strategy provides a powerful approach for the quantitative detection of microRNAs in vivo, providing a reference for the clinical treatment of cancer.

Scheme 1. Quantitative detection of the absolute miR-21 concentration via PA and ratiometric SERS imaging in vivo. (a) Schematic illustration of the sensing principle of the core–satellite nanoprobe for PA and ratiometric SERS imaging quantitative detection of miR-21 in vivo. In the presence of miR-21, the AuNNP@AuNP core–satellite showed responsively enhanced PA signals at 820 nm and enhanced SERS₁ signals at 2226 cm⁻¹, while the SERS₂ signals at 1378 cm⁻¹ remained stable, resulting in enhanced ratiometric SERS imaging. (b) The AuNNP probe/AuNP probe self-assembled into the AuNNP@AuNP core–satellite nanoprobe with enhanced PA signals and ratiometric SERS signals for accurately quantifying the absolute concentration of miR-21 at any sites of tumor and tracking its distribution.

Fig. 1. Characterization of AuNNP probe/AuNP probe controllable assemblies. (a) Schematic diagram of the AuNNP probe/AuNP probe self-assembled into AuNNP@AuNP core–satellite nanoprobes in the presence of miR-21. AuNP/MBN and AuNNP/NAT were modified with SH-DNA₁ and SH-DNA₂, which were partly complemented with miR-21, respectively. Representative transmission electron microscopy (TEM) images (b–i), dark field microscope (DFM) images and magnified DFM images (inset) (j–q) of AuNNP probe/AuNP probe with different concentrations of miR-21: (b and j) 0 pM, (c and k) 25 pM, (d and l) 50 pM, (e and m) 75 pM, (f and n) 100 pM, (g and o) 125 pM, (h and p) 150 pM, and (i and q) 175 pM. The scattering color of AuNNP probe/AuNP probe changed from green spots to orange-yellow spots and then to red spots with the increased concentration of miR-21.

Fig. 2. The formation process of AuNNP probe/AuNP probe controllable assemblies. (a–f) TEM images of different incubation times of AuNNP probe/AuNP probe: (a) 1 h, (b) 2 h, (c) 3 h, (d) 4 h, (e) 5 h and (f) 6 h. (g) The plot of the I₂₂₂₆/I₁₃₇₈ ratio of the AuNNP probe/AuNP probe at different incubation times (from 1 to 6 h). UV-vis spectra (h) and DLS (i) of the AuNNP probe/AuNP probe in the presence of miR-21 or in the absence of miR-21.

Fig. 3. The sensitivity and specificity of the AuNNP probe/AuNP probe for detecting the miR-21 concentration in vitro. SERS spectra (a), the plot of the I₂₂₂₆/I₁₃₇₈ ratio (b) and the standard calibration curve (c) of the AuNNP probe/AuNP probe that responded to different concentrations of miR-21 (from 0 to 175 pM). (d) The ratiometric SERS signals of the AuNNP probe/AuNP probe for mismatched sequences of miR-21 (mismatch-1), miR-144, miR-155, miR-429, Let-7a and miR-21 in vitro (concentration of 175 pM). (e) Linear response of the average PA intensity at 820 nm and representative PA images (inset) of the AuNNP probe/AuNP probe toward miR-21 in the range of 0 to 1 nM. (f) The average PA intensity at 820 nm of the AuNNP probe/AuNP probe for mismatch-1, miR-144, miR-155, miR-429, Let-7a and miR-21 in vitro (concentration of 1 nM). Statistical significance was calculated by the t-test (n = 3): **p < 0.01 and ***p < 0.001.

Fig. 4. SERS-based ratiometric strategy for monitoring intracellular miR-21 distribution and assembly behavior of AuNNP@AuNP core–satellite nanoprobes. (a) Schematic illustration of the ratiometric detection of miR-21 using the AuNNP probe/AuNP probe in MCF-7 cells or LO2 cells. As the AuNNP probe/AuNP probe endocytosed into MCF-7 cells, overexpressed miR-21 activated the AuNNP probe/AuNP probe self-assembly to form a core–satellite nanostructure, resulting in an enhanced Raman intensity from MBN on the surface of the AuNP probe, whereas the internal standard signal from NAT in the nanogap of the AuNNP probe remained stable. Intracellular DFM images (b), SERS mapping images at 1378 and 2226 cm⁻¹ and ratiometric (I₂₂₂₆/I₁₃₇₈) images (c), and bio-TEM images and magnified bio-TEM images (inset) (d) of MCF-7 cells and LO2 cells after incubation with the AuNNP probe/AuNP probe (2 mg mL⁻¹) for 8 h, 12 h, 18 h, and 24 h. The untreated cells are taken as blanks for DFM (40× objective, 700 ms integration time). The scale bars are (b) 20 μm, (c) 10 μm, and (d) 1 μm.

Fig. 5. PA imaging for tracking the distribution of miR-21 in Balb/c nude mice bearing MCF-7 tumors using the AuNNP probe/AuNP probe. (a) The photograph of Balb/c nude mice bearing MCF-7 tumors for PA imaging. Representative PA images (b) and quantified PA intensities (c) of Balb/c nude mice bearing MCF-7 tumors as a function of post-injection time (0.5 h, 4 h, 8 h, 12 h, 24 h, and 36 h) of the AuNNP probe/AuNP probe or phosphate buffer solution (PBS).

Fig. 6. Ratiometric SERS imaging quantitative detection of miR-21 in Balb/c nude mice bearing MCF-7 tumors using the AuNNP probe/AuNP probe. (a) Bright field images and SERS mapping images at 1378 and 2226 cm⁻¹ and I₂₂₂₆/I₁₃₇₈ of the AuNNP probe/AuNP probe treated Balb/c nude mice bearing MCF-7 tumors or normal tissue (muscle). The scale bar is 10 μm. (b) The average relative miR-21 expression levels in MCF-7 tumor tissue and normal tissue obtained via ratiometric SERS imaging and qRT-PCR. Error bars represent standard deviation (n = 3). (c) The corresponding miR-21 mapping at representative MCF-7 tumor sites in mice. (d) The value of the absolute miR-21 concentration from different tumor regions in (c), and the overall average value of the absolute miR-21 concentration. Error bars represent standard deviation (n = 6). (e) The corresponding absolute concentration of miR-21 in MCF-7 tumor tissue at 8 h, 12 h, and 18 h post-injection of the AuNNP probe/AuNP probe. Error bars represent standard deviation (n = 3).