At present many methods have been applied for

At present, many methods have been applied for multiple detection. For example, gap 27 [[9], [10], [11]] is frequently used to detect multiple targets because of its exquisite sensitivity and specificity, but it is difficult to design experiments to test short-length oligonucleotides. Electrochemical sensors [[12], [13], [14]] have been widely applied for sensitive and simultaneous detection of multiple biomarkers, because of time-saving, simple, and inexpensive analysis. However, their use in various applications has not expanded due to their instability and the difficulty in modification of their electrodes, which limit development. Surface-enhanced Raman spectroscopy (SERS) [[15], [16], [17], [18]] is feasible for simultaneous detection, because samples can be rapidly detected with high accuracy and without the necessity of sample pretreatment. However, it would be necessary for SERS probes to be combined with metal nanoparticles for this type of detection, leading to complicated synthetic steps. In addition, fluorescent methods, with the advantage of simple operation, stability, and fast detection, have also been developed for simultaneous detection based on molecular beacon and fluorescence quenching methods [[19], [20], [21], [22], [23]]. For example, Wang et al. has developed few-layer graphdiyne nanosheets that are used as novel sensing platforms for a variety of fluorophores for real-time detection of DNA with low background and high signal-to-noise ratio [24]. Nevertheless, experiments are complex and difficult to perform because of the synthesis of quencher and modification of fluorophores. Zhu's group has synthesized a silver nanocluster beacon that can be activated as a stimuli-responsive versatile platform for multiplex target detection [25]. However, some limits, such as requiring additional quencher and cumbersome modification, also exist with these methods. To address these difficulties mentioned above, we sought to design a label-free and quencher-free simultaneous detection strategy for multiple targets. In our work, rapid, quencher-free, and label-free simultaneous detection of multiple targets is proposed in combination with G-quadruplexes and DNA-templated silver nanoclusters (AgNCs). Genes of influenza A virus subtypes H5N1 and H1N1 were chosen as the models in this study for simultaneous detection. First, thioflavin T (ThT), which is a water-soluble benzothiazole salt fluorescent dye that is cell-permeable with superior selectivity for G-quadruplex [26], was selected for label-free detection of H5N1. A nanocluster dimer produced by two closely split AgNCs [27] was selected for label-free detection of H1N1. Next, with the H5N1 addition, only the G-quadruplex/ThT duplex was formed for specific and enhanced fluorescence response. In addition, an increase in the fluorescence intensity of AgNCs only occurred with the presence of H1N1 because dark AgNCs became bright nanocluster dimers. Then, with the addition of H5N1 and H1N1, the probe with two capitated recognized regions was hybridized with targets. Additionally, a G-quadruplex/ThT duplex and nanocluster dimer was formed, and the corresponding fluorescence was produced. Moreover, this label-free simultaneous detection possessed good sensitivity with a detection limit of 0.45 nM (H5N1) and 10 nM (H1N1). MicroRNA-141 (miR-141) and microRNA-21 (miR-21) are both biomarkers of prostate cancer and indicators for identifying the stages of prostate cancer [19]. MiR-141 is significantly elevated in advanced prostate cancer, but its expression is normal in the early stages of the cancer. However, miR-21 is overexpressed in the early stage, but not in advanced prostate cancer. The versatile label-free method described above was also performed for the simultaneous detection of miR-141 and miR-21 with great sensitivity and selectivity. This approach also had various advantages, such as rapid detection in 40 min, low cost without the need for labeling procedures, and process simplicity without requiring large precision instruments. This simultaneous detection method would also be able to detect targets in biological fluids, and there is great potential application for its use in clinical diagnostics and biomedical research.