Search

A sensitive ratiometric fluorescence probe based on hybridization chain reaction (HCR) was constructed for sensitive detection of miRNA-21 by using G-triplex and silver nanocluster pairs (AgNC pairs) as an enzyme-free and label-free signal output group. miRNA-21 was used as the primer for the hybridization chain reaction of molecular beacon 1 (MB1) containing the locked G-triplex sequence and molecular beacon 2 (MB2) with intact AgNC pairs at the 5′ and 3′ end activation. The double-stranded product was obtained along with the opening of the G-triplex and the separation of the AgNC pairs. A detection limit of 67 pM and a linear detection range of 0.1–300 nM were obtained for miRNA-21 determination. The proposed strategy enabled the monitoring of miRNA-21 levels in at least three cell lines, indicating that it provided new ideas for detecting miRNA in real samples. [ABSTRACT FROM AUTHOR]

An enzyme-free resonance light scattering (RLS) method is described for the determination of microRNA-122. A guanine nanowire (G-wire) is used that consists of a predesigned DNA1 and a G-quadruplex sequence DNA2. These hybridize with microRNA-122 and partially hybridize with DNA2. After formation of stable double strands with DNA1, DNA2 is released. On addition of K+ and Mg2+ ions, the G-quadruplex sequences undergo self-assembly to form long filamentous G-wires. This increases the intensity of RLS. A 6.1 pM detection limit was obtained, and the linear response covers the 50 pM to 300 nM microRNA concentration range. The method was successfully applied to the quantitation of microRNA-122 in hepatocellular carcinoma cell lysates. Conceivably, this assay can be extended to other RLS methods for biomarker detection by simply changing the sequence of DNA1. [ABSTRACT FROM AUTHOR]

A simple, highly selective, and label-free microRNA (miRNA) detection method based on l -alanine-reduced graphene oxide fluorescence quenching with a Y-shaped probe is proposed. The Y-shaped probe was synthesized by silver nitrate and a cytosine-rich molecular beacon (MB) in two terminals through sodium borohydride reduction, which generated a stronger fluorescent signal than ordinary DNA-templated silver nanoclusters (AgNCs). Meanwhile, the Y-shaped probe contained a single-stranded loop structure, which could be superbly adsorbed onto the surface of reduced graphene oxide (RGO) via π–π stacking interaction, and this special structure of the probe was designed to improve its sensitivity and selectivity. In addition, the quenching capacities of graphene oxide (GO) and RGO were compared in this research. The strong interaction between nucleobases of the loop structure and RGO nanosheet made the MB-AgNCs-RGO system exhibit minimal background fluorescence. In the presence of miRNA-21, the loop structure of the Y-shaped probe can hybridize with target miRNA-21; the molecular beacon encapsulated probe is far away from RGO surface and produces a detectable signal. The MB-AgNCs based approach provides a label-free avenue to detect miRNA with high selectivity and good reproducibility, which has a promising application in early clinical diagnosis and biomedical research. [ABSTRACT FROM AUTHOR]

A molecular beacons (MBs) loaded on molybdenum disulfide (MoS 2) nanosheets as fluorescence probes for sensitive and versatile detection of microRNAs (miRNAs) through hybridization chain reaction (HCR) has been designed. MoS 2 was used as a adsorbent to capture the MBs and a selective fluorescence quencher to reduce the background signal. In the absence of miRNAs, HCR could not be triggered due to the stability of MB probes. The probes attached to the MoS 2 surface, efficiently quenching fluorescence of the G-quadruplex/Thioflavin T. However, the presence of target miRNAs triggers the HCR process to generate large amount of HCR products. Meanwhile, the HCR products of long nanowires chain with abundant G-quadruplexes could not be adsorbed on the surface of MoS 2 , and therefore detach from the MoS 2. Consequently, Thioflavin T could be embedded in G-quadruplexes and produced strong fluorescence signal. This fluorescence emission signal could achieve detection of miRNA as low as 4.2 pM and a wide linear ranges from 0.1 to 100 nM. In addition, a versatile fluorescence probe has been developed for detection of miRNA-21 by changing the miRNA-recognition domain of MB. Thus, the fluorescent probe would be a potential alternative tool for biomedical research and clinical molecular diagnostics. MB1 and MB2 hairpins DNA can absorb onto MoS 2 nanosheets without target miRNA. In the presence of target, it can opened the hairpin structure of MB1 and caused the rest sequence of MB1 to bind with parts of MB2. Similarly, after MB2 was opened, the exposed parts of MB1 hybridized with the complementary sequences of another MB2. Such HCRs can generate a long MB1-MB2 duplex chain which desorbed from MoS 2 nanosheets and the fluorescence intensity of G-quadruplex/ThT was recoveried. Image 1 • A low-background MoS 2 nanosheets-loaded molecular beacon probe with signal amplification by HCR technology for different miRNAs detection. • This lable-free and enzyme-free, high selectivity, cost-saving miRNAs detection assay with the limit of detection as low as 4.2 pM. • MiRNAs detection in cell lysates with satisfactory results. [ABSTRACT FROM AUTHOR]

Abstract A rapid, label-free, and multifunctional fluorescent probe for simultaneous detection of multiple targets was fabricated based on G-quadruplexes and DNA-templated silver nanoclusters (AgNCs). In this work, a probe with two capitated recognized regions was coupled with a locked G-quadruplex at the 5′-terminus and dark AgNC at the 3′-terminus. Upon the addition of the virus subtype H5N1 gene or microRNA-141, only the sequence of the G-quadruplex was released and bound with Thioflavin T (ThT) for a specific fluorescent response. On the contrary, with the presence of the influenza virus subtype H1N1 gene or microRNA-21, the fluorescence intensity was enhanced because of two split AgNCs approaching closely to produce a nanocluster dimer. Subsequently, with multiple target addition, fluorescence signals were produced for both G-quadruplexes and AgNCs. Moreover, this single and duplex detection for virus DNAs and microRNAs, which provides a versatile platform for different targets, was sufficiently sensitive for the expected detection limit, and still possessed unique selectivity with similar oligonucleotides. The simultaneous detection of targets in biological fluids indicated that there is great opportunity for this strategy to be further applied in biomedical research and clinical diagnosis. Graphical abstract Image 1 Highlights • A rapid, label-free and multifunctional fluorescent probe was fabricated to overcome cumbersome labeling procedures. • The system based on G-quadruplexes and DNA-templated silver nanoclusters (AgNCs) was designed for improving the sensitivity for simultaneous detection of virus DNA and microRNA. • The proposed strategy exhibited special selectivity in similar oligonucleotides and could be well applied in biological fluids. • The detection limit for H5N1 and H1N1 was estimated to be 0.45 nM and 10 nM respectively, and for miRNA-141 and miRNA-21was 1 nM and 10 nM respectively. [ABSTRACT FROM AUTHOR]

An efficient dual-amplification system based on catalyzed hairpin assembly (CHA) amplification and hybridization chain reaction (HCR) circuits is developed for G-quadruplex-based and non-enzymatic fluorescence detection of microRNA. Four hairpin probes, namely, H1, H2, H3, and H4, are excellently designed to coexist metastably without a target. Upon the addition of the target, H1 recognizes the target and releases the G-quadruplexes. Meanwhile, H2 fully hybridizes with H1 to form stable H1/H2 duplex that leads to the replacement of the target for participating in another cycle. Moreover, the newly formed product of CHA cycles, H1/H2 duplex, hybridizes with H3 and frees the trigger to open HCR circuits, resulting in the cross-opening of H3 and H4 and self-assembling into a long DNA nanowire with abundant G-quadruplexes. Consequently, many G-quadruplex structures are formed and further combined with thioflavin T for significant fluorescence enhancement. The proposed system can detect miRNA in a wide concentration range with the detection limit as low as 36 fM and can exhibit special selectivity in homologous miRNA sequences. Moreover, this detection assay can be well applied in cancer cell samples, which possesses enormous potential to show novel perspectives in the analysis of biomarkers, clinical diagnostics, and biomedical applications. [ABSTRACT FROM AUTHOR]

Based on 2-aminopurine (2-AP) probe in conjunction with a G-quadruplex structure and signal amplification technique, a simple and highly sensitive fluorescence sensor for detecting microRNA (miRNA) is developed for high signal-to-background ratio and wide linear range. The proposed sensor contains two hairpins DNA: H1 and H2. H1 is labeled by 2-AP incorporated into a G-rich sequence. Upon the addition of a target miRNA, H1 is unfolded and forms DNA/RNA complexes that contain a G-quadruplex, thereby significantly enhancing 2-AP fluorescence due to the protection provided by the G-quadruplex. Subsequently, H2 can displace the miRNA from the DNA/RNA complexes and induce signal amplification, resulting in further enhanced fluorescence intensity. Hence, the sensor is highly sensitive and its low limit of detection (L.O.D.) can reach as low as 1.48 pM. Furthermore, the proposed sensor is used to detect overexpressed miRNA-21 from human breast cancer cell lysate. The result demonstrates the potential of the proposed sensor to monitor different miRNA biomarkers for the early diagnosis of various cancers. [ABSTRACT FROM AUTHOR]

An interference-free and label-free sensing platform was developed for the highly sensitive detection of microRNA-21 (miRNA-21) in vitro by magnetic silicon microsphere (MNP)-reduced graphene oxide (rGO)-based sandwich probe. In this method, DNA capture probes (P 1 ) were connected with MNPs at the 5′ end and hybridized with completely complementary target miRNA. Subsequently, rGO was retained and induced the fluorescence quenching in the supernatant. Through the magnetic separation, the supernatant environment was simplified and the interference to analytical signal was eliminated. When DNA capture probe-modified magnetic silicon microspheres (MNP-P 1 ) were adsorbed through rGO in the absence of a target and formed a sandwich structure, the formed nanostructure was easily removed from the solution by a magnetic field and the fluorescence intensity was maximally recovered. This proposed strategy, which both overcame the expensive and cumbersome fluorescent labeling, and eliminated interference to analytical signal for guaranteeing high signal-to-background ratio, exhibited high sensitivity with a detection limit as low as 0.098 nM and special selectivity toward miRNA-21. The method was potentially applicable for not only detection of miRNA-21 but also various biomarker analyses just by changing capture probes. [ABSTRACT FROM AUTHOR]

A quencher-free and enzyme-free fluorescent sensor was proposed to simply and sensitively detect miRNA via the target catalyzed hairpin assembly (CHA) signal amplification in combination with 2-aminopurine (2-AP) molecular beacon (MBs). This sensor contains two DNA hairpins termed as H1 and H2. H1 labeled by 2-AP needs no quenchers because 2-AP can be quenched through its stacking interaction with the adjacent bases. H2 is partially complementary to H1. In the presence of the target microRNA (miRNA), H1 is unfolded and produces the DNA/RNA complexes, enhancing the fluorescent signal. Then, the RNA of the DNA/RNA complexes can be displaced by H2 and the free miRNA can interact with another H1, resulting in the significant fluorescence enhancement of the system. This signal amplification process is enzyme-free, making the sensor more simple and cost effective. The detection limit of this sensor could be as low as 3.5 pM. We further applied this assay to monitor the overexpressed miRNA-21 from human breast cancer cells to confirm its applicability. The proposed sensor could be used as a simple and sensitive platform for target miRNA detection, holding great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers. [ABSTRACT FROM AUTHOR]

The simultaneous detection of two important microRNAs (miRNAs) can potentially evaluate pathological states. The simultaneous detection of miRNA-26a and miRNA-122 is proposed in this study using a cleverly designed single-labeled, dual-functional fluorescent probe with a 2-aminopurine as a fluorophore, which is a G-quadruplex single-stranded DNA. The probe can partly complement cDNA-1 and cDNA-2. cDNA-1 and cDNA-2 are complementary strands of miRNA-26a and miRNA-122, respectively. When the target miRNAs are added simultaneously, these cDNA (cDNA-1 and cDNA-2) can be competed off from the cDNA∖probe duplex to form a cDNA∖RNA heteroduplex. The probe is released and the fluorescent signal is increased. The detection limits of miRNA-26a and miRNA-122 are both 2.5 nM, and their wide linear which ranges from 2.5 to 500 nM are achieved using the assay. This method has potential practical applications. [ABSTRACT FROM AUTHOR]

We report a rapid and facile resonance light scattering (RLS) technique that utilizes CdTe quantum dots (CdTe QDs) probe to detect microRNA-122. The RLS sensor is ingeniously designed with P1 and P2, two cDNA sequence probes with partially complementary sequences to miRNA-122. The amine-modified P1 and P2 are coupled to the surface of QDs to form functional QDs-P1 and QDs-P2 conjugates, which are collectively referred to as QDs-P. The cDNAs hybridize with the target miRNA to rapidly induce the self-assembly of QDs probe and change RLS intensity. The proposed technique can detect miRNA-122 within 40 min. RLS intensity is enhanced in proportion with miRNA-122 concentrations of 0.16–4.80 nM and has a low detection limit of 9.4 pM. In addition, the assay satisfactorily detects miRNAs in human serum samples. Thus, the assay has considerable potential for the analysis of other interesting tumor makers. [ABSTRACT FROM AUTHOR]

Herein, a metal-organic framework (UiO-66-NH 2) with two functions (intrinsic fluorescence and fluorescence quenching ability) is designed to establish a ratiometric fluorescent platform for high-performance miRNA detection. The use of a fluorescent organic ligand endows the MOF material with a strong intrinsic fluorescence at 440 nm. In the presence of target miRNA, the fluorescence signal of the FAM is restored with the triple helix molecular beacons bind to the target. Using the I FAM /I MOF signal as the output, the prepared ratiometric probe was able to eliminate disturbance caused by the sensing environment. Under the optimal reaction conditions, including buffer pH of 7.4, temperature of 37 °C, and response time of 1 h, the best detection results can be obtained. The ratiometric fluorescence probe presented showed good sensitivity and selectivity for detecting miRNA-203 and the limit of detection was 400 pM with a wide linear range from 1 nM to 160 nM. In addition, this method was applied to diluted human serum and cell lysates, and good detection effect was realized. In the absence of miRNA-203, the FAM-modified P2 was closed to the UiO-66-NH 2 due to the formation of triple helix molecular beacon, so that the fluorescence of FAM was quenched. In the presence of miRNA-203, the P2 was hybridized with miR-203 and release the long strand DNA P1. Thus, FAM went away from UiO-66-NH 2 , resulting in significant fluorescence enhancement. [Display omitted] • UiO-66-NH 2 can apply to miRNA-203 detection at picomolar level as a new fluorescence quencher and fluorescence output signal. • This UiO-66-NH 2 has a larger surface area, unique resistance to enzyme digestion, and efficient endocytosis. • The proposed fluorescent probe can be further applied to actual samples such as cell lysates and human serum samples. [ABSTRACT FROM AUTHOR]