Simultaneous and ultrasensitive detection of multiple microRNAs by single-molecule fluorescence imaging

Abstract

Cell status changes are typically accompanied by the simultaneous changes of multiple microRNA (miRNA) levels. Thus, simultaneous and ultrasensitive detection of multiple miRNA biomarkers shows great promise in early cancer diagnosis. Herein, a facile single-molecule fluorescence imaging assay was proposed for the simultaneous and ultrasensitive detection of multiple miRNAs using only one capture anti-DNA/RNA antibody (S9.6 antibody). Two complementary DNAs (cDNAs) designed to hybridize with miRNA-21 and miRNA-122 were labelled with Cy3 (cDNA1) and Cy5 (cDNA2) dyes at their 5'-ends, respectively. After hybridization, both miRNA-21/cDNA1 and miRNA-122/cDNA2 complexes were captured by S9.6 antibodies pre-modified on a coverslip surface. Subsequently, the Cy3 and Cy5 dyes on the coverslip surface were imaged by the single-molecule fluorescence setup. The amount of miRNA-21 and miRNA-122 was quantified by counting the image spots from the Cy3 and Cy5 dye molecules in the green and red channels, respectively. The proposed assay displayed high specificity and sensitivity for singlet miRNA detection both with a detection limit of 5 fM and for multiple miRNA detection both with a detection limit of 20 fM. Moreover, it was also demonstrated that the assay could be used to detect multiple miRNAs simultaneously in human hepatocellular cancer cells (HepG2 cells). The proposed assay provides a novel biosensing platform for the ultrasensitive and simple detection of multiple miRNA expressions and shows great prospects for early cancer diagnosis.

Scheme 1. Schematic illustration of single-molecule fluorescence imaging assay for the simultaneous detection of multiple miRNAs.

Fig. 1. Simultaneous detection of multiple miRNAs by the single-molecule imaging assay. The concentration of both miRNA-21 and miRNA-122 is 500 pM. Scale bar represents 5 μm.

Fig. 2. Influence of the binding time of the DNA/RNA complex and S9.6 antibody. (A) miRNA-21; (B) miRNA-122. Error bars represent the standard deviation of three experiments.

Fig. 3. Single-molecule fluorescence imaging of various concentrations of singlet miRNA, (A) miRNA-21, and (C) miRNA-122. Standard curve of the number of image spots as a function of singlet miRNA concentrations for (B) miRNA-21 and (D) miRNA-122, respectively. Error bars represent the standard deviation of three experiments. Scale bars represent 5 μm.

Fig. 4. (A) Single-molecule fluorescence imaging for simultaneous detection of multiple miRNAs. (B) Standard curve of the number of image spots from Cy3 as a function of miRNA-21 concentration. (C) Standard curve of the number of image spots from Cy5 as a function of miRNA-122 concentration. Error bars represent the standard deviation of three experiments. Scale bars represent 5 μm.

Fig. 5. Specificity assessment of the single-molecule imaging assay for the measurement of single-base mismatched miRNAs (SM miRNA), three-base mismatched miRNAs (TM miRNA), and noncomplementary miRNAs (miRNA-143, miRNA-16, and miRNA-141).

Fig. 6. (A) Linear relationship between Cy3 image spots and the logarithm of the number of HepG2 cells; (B) linear relationship between Cy5 image spots and the logarithm of the number of HepG2 cells. Error bars represent the standard deviation of three experiments.