MicroRNAs (miRNAs) are sorts of small single stranded noncoding RNAs and its length are approximately 21 ~ 25 nucleotides [1–3]. Since the dysregulation of miRNAs is closely related to the occurrence and prognosis of tumor, the detection of circulating miRNAs in serum can be used as credible biomarkers for tumor liquid biopsy [4, 5]. For instance, monitoring the circulating miRNA-150 in serum could provide reliable clinical assays for diagnosis early non-small cell lung cancer (NSCLC) . Thus, the sensitive and specific detection of miRNAs is considered to be a meaningful strategy for early cancer diagnosis.
Currently, the detection of miRNAs is normally performed by traditional techniques such as northern blotting, microarray and reverse transcription-polymerase chain reaction (RT-PCR) [7–9]. Although these methods are reliable, they are hardly applied to clinic due to the disadvantages of complicated operations, high cost, unstable results and required well-trained staff. Certainly, other various techniques have been developed, including electrochemical sensor, surface plasmon resonance (SPR), surface-enhanced Raman scattering (SERS), colorimetry and fluorescence [10–14]. Among the various techniques, fluorescence detection platform has emerged as an excellent alternative for the detection, quantification and characterization of target [15–17]. Organic fluorescent dye-labeled DNA probe and fluorescent technique have been widely employed for ultrasensitive quantification of DNA, miRNA, proteins, and others because of the easily commercialized synthesis [18–20]. To upgrade the assay sensitivity, most of these determinations were carried out assist with signal amplification strategies, including rolling circle amplification (RCA), exponential amplification reaction (EXPAR), catalytic hairpin assembly (CHA), entropy driven catalytic reaction, enzyme-assisted signal amplification and materials, etc [21–26]. However, these amplified methods also suffered from various drawbacks and limitations in practice, such as long time and high-budget needed, reaction condition rigorous, not well met the demand of universal simple and rapid medical analysis, especially for point-of-care testing (POCT). Therefore, it is expected to establish one kind of simpler method for target directly detection without the help of any amplification strategy.
One possible solution is that the allosteric spherical nanoprobe with high reaction cross section and showing high binding capacity and specificity for the target miRNA. Up to now, the hairpin probe has always been attracted particular interest as the simplest, prototypical system. Tyagi and Kramer were the pioneers to study hairpin shaped molecular probe . The nature of molecular beacon is single-stranded DNA molecule, which contains a stem-and-loop structure and a pair of fluorophore and quencher group. The loop is design to hybridize with its complete complementary target. And the stem is the result of two complementary arm sequences through annealing. Due to the proximity of a pair of fluorophore and quencher group, the fluorescence was quenched. So, when the loop recognizes the perfectly target, the structure of hairpin changes into a more stable formation of DNA duplex and forces the stem apart, resulting in the leakage of fluorescence. To date, various molecular probe-based detection methods have been proposed in a large scale of applications. For example, the detection of DNA damage, the monitoring of DNA movement, biological small molecule detection, and miRNA detection in living cells [28–33]. Nevertheless, molecular probe-based nucleic acids detection is also generally on the basis of the above-mentioned amplification techniques.
Here, we design a unique allosteric spherical nanoprobe based on the traditional molecular beacon idea. Fluorescent groups and quenching groups are respectively marked at the end-to-end joint of the two hairpin structures. Numerous dual-hairpin structures are modified on the surface of magnetic nanoparticles. So, the nanoprobe was endowed with enrichment capacity and reaction cross section so as to be easier to react with the target. When the target is existing, the closed dual-hairpin spherical nanoprobe is opened to active “Y” structure, leading to significant fluorescence leakage. Following that, the results of fluorescence were recorded on multipoint fluorescence scanning microarray. The manner of fluorescence readout is different from traditional fluorescent scanner, the microarray device is more portable and compact, especially for POCT application. In this way, the miRNA-150 detection is determined by the fluorescence changes in the allosteric spherical nanoprobe upon binding with the target. Due to the inherent fluorescence signal transduction mechanism, the dual-hairpin spherical structure functions as a sensitive probe with a high signal-to-background ratio. Meanwhile, this allosteric spherical nanoprobe has high hybridization specificity because of its loop-stem structure, which can easily discriminate the complementary from single-mutation target miRNA.