MNAzymes and gold nanoparticles as isothermal signal amplification strategy for visual detection of miRNA

Abstract

MicroRNAs (miRNAs) represent a class of small noncoding RNAs that are considered a novel emerging class of disease biomarkers in a variety of afflictions. Sensitive detection of miRNA is typically achieved using hybridization-based methods coupled with genetic amplification techniques. Although their sensitivity has improved, amplification techniques often present erroneous results due to their complexity. In addition, the use of these techniques is usually linked to the application of protein enzymes, the activity of which is dependent on the temperature and pH of the medium. To address these drawbacks, an alternative genetic enzyme for the highly sensitive detection of miRNAs is proposed in this work. Multicomponent nucleic acid enzymes (MNAzymes), coupled with the use of DNA-functionalized gold nanoparticles (AuNPs), were used in this study to develop an isothermal signal amplification strategy for visual genetic detection. miR146a, a biomarker of bovine mastitis present in milk, was selected as a model analyte. The developed methodology is easily carried out in 80 min at 50 °C, generating a low visual limit of detection of 250 pM based on the observation of a color change. The methodology was successfully applied to the detection of miR146a in raw cow milk samples.

Keywords: AuNPs; Color detection; MNAzymes; Signal amplification; microRNA.

Fig. 1

Scheme for the detection of a microRNA target

Fig. 2

Optimizing the concentrations of [DNA-linker] and [MgCl₂]. SPR wavelength and TLC spots of AuNPs at 10 mM, 15 mM, 20 mM, and 30 mM MgCl₂ concentrations; and 0 nM, 20 nM, 30 nM, 40 nM, 45 nM, 50 nM, 60 nM, and 70 nM DNA-linker concentrations. All the experiments were performed in triplicate (n = 3)

Fig. 3

A Image of the AuNPs deposited onto the TLC plates in the presence of miR146a: For target concentrations higher than 100 pM, the color observed is pink, whereas for the blank, it is dark purple. B Representation of the variation in the SPR wavelength versus the miR146a concentration: 0 pM, 50 pM, 100 pM, 250 pM, 500 pM, 1000 pM, 2500 pM, and 5000 pM. All measurements were performed in quintuplicate (n = 5)

Fig. 4

Detection of short RNA sequences after changing one and two bases (underlined in black) was changed from miR146a: modification of 1 base (1U and 1C) and two bases (2GC). These studies were carried out in triplicate (n = 3)

Fig. 5

Image of the AuNPs deposited onto the TLC plates in the presence of miR146a in milk samples. Increasing miR146a standard additions of 0 pM, 50 pM, 100 pM, 250 pM, 500 pM, 1000 pM, 2500 pM, and 5000 pM were spiked into raw milk samples from healthy cows. At 250 pM of target concentration, the color observed is pink, while dark purple is observed for the blank. The standard additions and detection assays were carried out in triplicate (n = 3)