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Because rapid, convenient, and selective methods for HIV detection are urgently needed, herein, a simple label-free and enzyme-free strategy is constructed for sensitive fluorescence detection of HIV DNA using the fluorescent intercalating dye thioflavin T (THT) as the detection signal source. This strategy utilizes a hairpin DNA sequence (H1) and two assistant strands. H1 is wisely designed with a G-quadruplex sequence in the stem. Target DNA, when present in solution, will hybridize with H1 to form H1/target duplexes and release the G-quadruplexes. Additionally, the assistant probes hybridize with the unfolded H1 to form a stable DNA double strand, resulting in the displacement of the target to participate in another similar reaction cycle. Consequently, many G-quadruplex structures are generated, leading to a significantly amplified fluorescence signal of THT. The linear range is from 0.1 nM to 50.0 nM with a limit of detection of 13 pM. Results can be achieved within 40 min, because the cyclic amplification involves only one DNA hairpin and two auxiliary chains. Furthermore, this platform exhibited good selectivity with one base mismatch or other DNA sequences. This strategy could be used as a simple, sensitive, and selective tool to detect other DNA biomarkers. [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 widely applicably method is described for fluorometric determination of targets such as microRNA and viral DNA. It is making use of a Mg(II)-dependent DNAzyme and a G-quadruplex. In the absence of analyte, an inactive DNAzyme is formed by the hybridization of split DNAzymes and substrate. On addition of target analyte, the end of each strand of the split DNAzymes bind the analyte. This leads to the generation of an active DNAzyme. In the presence of Mg(II), the activated DNAzyme is formed and can cleave the substrate strand. Hence, the caged G-quadruplex sequences will be released. These released G-quadruplexes combine with thioflavin T to generate a G-quadruplex/thioflavin T complex and thereby cause amplified fluorescence. The method shows a 70 pM detection limit for H5N1 and works over a wide linear range 1 nM to 400 nM. Conceivably, this detection scheme has a wide scope in that it may be applied to other assays for microRNAs and DNAs by variation of the type of DNAzyme. [ABSTRACT FROM AUTHOR]

A DNA-templated copper nanoparticle (CuNP) probe has been developed for the determination of the human immunodeficiency virus oligonucleotide (HIV-DNA). The function of the probe relies on affinity binding-induced DNA hybridization associated with the use of double G-quadruplexes. Double-stranded DNA (dsDNA) with poly(AT-TA) bases was used as a template for synthesis of dsDNA-CuNPs. These have weak fluorescence. In the next step, two G-rich sequences that are linked to both sides of the ds-DNA are locked by HIV complementary DNA (cDNA). If HIV-DNA is introduced, it will hybridize with cDNA, thereby transforming the two G-rich sequences into G-quadruplexes. This enhances the fluorescence of the adjacent dsDNA-CuNPs. Fluorescence increases linearly in the 1 to 200 and 250-1000 nM HIV-DNA concentration range, and the detection limit is 13 pM. This enzyme-free fluorometric assay is time-saving, easily operated, and therefore has large potential in biosensing because it may be extended to various other DNA targets.Double-strand DNA-templated copper nanoparticles (DNA-CuNPs) have weak fluorescence. When Human Immunodeficiency Virus oligonucleotide (HIV-DNA) is added, it completely hybridized with HIV complementary DNA (cDNA). As a result, the two exposed G-rich sequences are transformed into G-quadruplexes, and an apparent increase in the fluorescence intensity can be observed. (AA: ascorbic acid). [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]

A facile and cost-effective fluorescence biosensor is constructed for the accurate and sensitive determination of DNA. The G-quadruplex-forming sequence in hairpin DNA sequence (H1) is originally locked. When target DNA is present, the hairpin structure of H1 can be unfolded and part of H1 hybridized with the target DNA to form a double-stranded DNA and a G-quadruplex. The hairpin DNA sequence (H2) hybridizes with the unfolded H1 and displaces the target DNA. Finally, the displaced target DNA again hybridizes with the H1 and initiates cycle amplification. Thus, the numerous G-quadruplexs at the H1 ends are formed, resulting in the fluorescent enhancement after binding with N-methyl mesoporphyrin IX (NMM). Lower background signal improves the accuracy of assay. The enzyme-free method can detect down to 40 pM and a linear range of 3 orders of magnitude. Moreover, this strategy has an ability to discriminate the target DNA from even single-base mismatched or other sequence. The proposed method has potential applications in DNA-based molecular diagnostics. [ABSTRACT FROM AUTHOR]

In the absence of H5N1 DNA, the fluorescence of 2-aminopurine (2-AP) was high at 363 nm and the fluorescence of thioflavin T (ThT) was low at 495 nm because the G-quadruplex (blue part in H1) forming sequences associated with ThT locked in the hairpin structures of H1. In the presence of H5N1 DNA, the target DNA binds with H1 and restores the fluorescence of ThT first. Then, H2 hybridizes with H1 and free the target DNA, which is used to trigger another reaction cycle to produce large amounts of G-quadruplex. The fluorescence of 2-AP remains unchanged and fluorescence of ThT was significantly enhanced. [Display omitted] • A 3D DNA walker is proposed for H5N1 DNA ratiometric determination with enzyme-free amplification. • A detection range of 0.2–20 nM was obtained and the detection limit of the DNA walker is 60 pM. • This DNA nanomachine has the advantage of simple, low-cost, sufficiently sensitive and higher versatility. The reliable and quantitative detection of virus DNA is highly desirable for the early diagnosis and clinical treatment of diseases. In this work, a three-dimensional (3D) DNA walker based on a non-enzyme-mediated nucleic acid cascade amplification reaction was constructed for the ratiometric detection of H5N1 DNA. When the DNA walker was developed by the surface modification of aminated silica microsphere (SiO 2 -NH 2) with H1 hairpin structures were activated in the presence of target DNA, the fluorescence of the H1-conjugated Thioflavin T (ThT) fluorophore (495 nm) was turned on, while the fluorescence of 2-aminopurine (2-AP) (363 nm) conjugated to the H2 hairpin remained unchanged. This strategy combines DNA walker powered by catalyzed hairpin assembly (CHA) reaction with proportional fluorescence signal output methods, which can effectively reduce the risk of generating false-positive signals. The developed DNA walker demonstrated excellent analytical sensitivity and specificity, with a detection limit of 60 pM. The DNA walker was also capable of selectively detecting H5N1 DNA in clinical blood serum samples, which demonstrated its potential for use in various clinical applications. [ABSTRACT FROM AUTHOR]

• A label-free resonance light scattering (RLS) aptasensor based on triple-helix molecular switch (THMS) is developed. • The designed aptasensor is label-free, low-cost, facile synthesis and simple preparation process. • The aptasensor holds a great promise for universal RLS sensing platform for detecting various targets just by changing the sequence of the THMS in the loop. In this work, a label-free resonance light scattering (RLS) biosensor based on triple-helix molecular switch (THMS) and G-quadruplex nanowires (G-wires) for HAV DNA and microRNA-31 detection was developed. The target can combine with the loop sequences of triple-helix, and opened the triple-helix structure as well as liberated the G-rich sequences which were self-assembled into G-wires in the presence of K + and Mg2+, causing an obvious enhancement of RLS intensity. Meanwhile, the RLS signal at 304 nm was linear with the logarithm of target concentration in the range of 50 pM to 300 nM, and the limit of detection is as low as 7.1 pM for HAV DNA and microRNA-31, respectively. Moreover, qualitative analysis of HAV DNA and microRNA-31 was successfully achieved in human serum samples and cancer cell lysates. The biosensor holds a great promise for universal RLS sensing platform for sensitive detection of various targets just by changing the sequence of the THMS in the loop and be a biosensing platform for early clinical diagnosis and biomedical research. Due to unique structure of triple-helix molecular switch (THMS), the G-rich sequences were locked by aptamer. The target is added to the solution containing the triple helix, target combined with the loop sequence of the triple helix, and opened the triple-helix structure to release the G-rich sequence. The free G-quadruplex sequences were self-assembled into G-quadruplex nanowires (G-wires) in the presence of Mg2+, causing an obvious enhancement of RLS intensity. Image, graphical abstract [ABSTRACT FROM AUTHOR]