Triple-helix Molecular Switch Research Articles

Detection of single nucleotide polymorphisms based on triple-helix molecular switch combined with invader assay

Single-nucleotide polymorphisms (SNPs) can be relevant to disease susceptibility, disease pathogenesis, and efficacy of specific drugs. Monitoring SNPs plays a significant role in disease susceptibility and individual response to treatment, wherein optimal care can be provided to individuals based on their unique genetic and disease profiles. In this work, we have formulated a rapid and sensitive fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced invader assay strategy for the detection of single nucleotide polymorphisms in the apolipoprotein E (APOE). The mutant DNA (MtDNA) of APOE gene hybridized with the THMS to form the three-dimensional invader structure over the SNP site. Subsequently, the specific structure would be recognized and cleaved by FEN1, leading to the recovery of FAM quenched by BHQ 1. Based on the high binding affinity of THMS to the target, our strategy could detect the MtDNA of AOPE gene without amplification at 0.27 nM. Furthermore, we achieved genotyping based on the difference in fluorescence signals and successfully applied this strategy to detect MtDNA of APOE gene in clinical samples, showing the great potential for monitoring various SNPs for biological research and disease diagnosis.

Mn2+-Triggered Swing-Arm Robot Strategy Using Anemone-Like Thi@AuPd@Cu-MOFs as Signaling Probes for the Detection of T-2 Toxin.

Herein, we synthesized anemone-like copper-based metal-organic frameworks (MOFs) loaded with gold-palladium nanoparticles (AuPd@Cu-MOFs) and polyethylenimine-reduced graphene oxide/gold-silver nanosheet composites (PEI-rGO/AuAg NSs) for the first time to construct the sensor and to detect T-2 toxin (T-2) using triple helix molecular switch (THMS) and signal amplification by swing-arm robot. The aptasensor used PEI-rGO/hexagonal AuAg NSs as the electrode modification materials and anemone-like AuPd@Cu-MOFs as the signal materials. The prepared PEI-rGO/hexagonal AuAg NSs had a large specific surface area, excellent electrical conductivity, and good stability, which successfully improved the electrochemical performance of the sensors. The AuPd@Cu-MOFs with high porosity provided a great deal of attachment sites for the signaling molecule thionine (Thi), thereby increasing the signal response. The aptasensor developed in this study demonstrated a remarkable detection limit of 0.054 fg mL-1 under optimized conditions. Furthermore, the successful detection of T-2 in real samples was achieved using the fabricated sensor. The simplicity of the THMS-based method, which entails modifying the aptamer sequence, allows for easy adaptation to different target analytes. Thus, the sensor holds immense potential for applications in quality supervision and food safety.

Triple-helix molecular switch-based light-addressable potentiometric aptasensor for the multi-channel highly sensitive label-free detection and spatiotemporal imaging of okadaic acid

Okadaic acid (OA) has attracted considerable attention in the fields of human health and public safety because it can cause severe virulence and is widely distributed. Therefore, it is of immediate significance to explore a novel method for highly sensitive, low-consumption, efficient, and convenient OA detection. In this study, a triple-helix molecular switch (THMS)-based light-addressable potentiometric aptasensor (LAPA) is proposed for the highly sensitive and selective label-free detection and spatiotemporal imaging of OA. In this method, a THMS containing an OA aptamer served as the recognition element, and a DNA microarray including a light-addressable potentiometric sensor was designed as the transducer. Meanwhile, poly (allylamine hydrochloride) was modified on the chip surface to enhance DNA absorption and signal detection. A detection platform was constructed to measure signals and scan spatiotemporal images. The realized biosensing system enabled the multichannel detection of OA in a linear range of 0.05–100 nM with low sample consumption, a detection limit of 0.03 nM, and a detection time of 27 min/channel and spatiotemporal imaging analysis with a resolution of 400 µm2/pixel and frame duration of 3.2 min/frame. Considering the technical developing of aptamer-based THMS and micro/nano biosensor, the proposed method will provide a promising approach for label-free detection and spatiotemporal analysis of OA with high sensitivity, specificity, and detection efficiency; low sample dosage; and simple operation.

A Novel Fluorescent Aptasensor for Arsenic(III) Detection Based on a Triple-Helix Molecular Switch.

A novel aptamer-based fluorescent-sensing platform with a triple-helix molecular switch (THMS) was proposed as a switch for detecting the arsenic(III) ion. The triple helix structure was prepared by binding a signal transduction probe and arsenic aptamer. Additionally, the signal transduction probe labeled with fluorophore (FAM) and quencher (BHQ1) was employed as a signal indicator. The proposed aptasensor is rapid, simple and sensitive, with a limit of detection of 69.95 nM. The decrease in peak fluorescence intensity shows a linear dependence, with the concentration of As(III) in the range of 0.1 µM to 2.5 µM. The whole detection process takes 30 min. Moreover, the THMS-based aptasensor was also successfully used to detect As(III) in a real sample of Huangpu River water with good recoveries. The aptamer-based THMS also presents distinct advantages in stability and selectivity. The proposed strategy developed herein can be extensively applied in the field of food inspection.

Attomolar analyte sensing technique for detection of Pb2+ and Hg2+ ions based on liquid crystal

Detecting the ultra-low abundance of heavy metals in real samples remains challenging and it requires the development of high-performance biosensing modalities. This work describes a novel strategy for design an ultra-sensitive liquid crystal (LC) aptasensor, in which the LCs reorientation induced by conformational changes of a triple helix molecular switch (THMS) structure is utilized for the quantitative detection of Pb2+ and Hg2+ ions. The developed aptasensor exhibits a wide linear range with the ultralow detection limits as low as 0.021 aM and 0.068 aM for Pb2+ and Hg2+, respectively. Besides, the results show that other analytes cause no interference. Noteworthy, this sensing strategy is easy to expand for other targets by altering the aptamer sequence without change of the triple-helix structure and can be used for multiple analyte detection. The developed aptasensor is promising powerful platform as a LC AttoSens for precision screening of hazardous residues in food analysis.

A sensitive and rapid sensing platform for ochratoxin A detection based on triple-helix molecular switch and CMC-EDC/NHS covalent immobilized paper

Ochratoxin A (OTA) is the most common mycotoxin contaminant that is widely distributed in food products. Herein, a sensitive and rapid sensing platform was constructed based on triple-helix molecular switch and CMC-EDC/NHS covalent immobilized paper. The aptamer-based triple-helix molecular switch (THMS) released signal transduction probe (STP) upon the addition of OTA, which was then enriched on the TR512-peptide functionalized paper modified with CMC-EDC/NHS chemistry. The sensing platform achieved a lower detection limit of 0.012 ng/mL with the linear range of 0.025–100 ng/mL. Notably, the sensing could be finished in less than 15.5 min without complex concentration process or expensive equipment. Additionally, the TR512-peptide based fusion protein was strongly-immobilized on the paper substrate via carbodiimide coupling, which prevented the fusion protein from leaching and enabled a large amount of solution molecules to bind with the fusion protein (up to 1 mL). Finally, a full-set of device was developed and applied in real samples with good accuracy. Taken together, the sensing platform integrating THMS with functionalized paper possesses practical application prospects in food samples and may offer a new protocol for the detection of multifarious target analytes by virtue of altering the aptamer sequence in THMS.

Nano-gold mediated aptasensor for colorimetric monitoring of acrylamide: Smartphone readout strategy for on-site food control

A simple aptasensor is embedded in the internal surface of a micropipette tip as the aptasensor substrate for the label-free monitoring of acrylamide. The aptasensor is based on the formation of the triple-helix molecular switch (THMS) structure of the DNA strands that induces the salt-enforced aggregation of gold nanoparticles (AuNPs). A smartphone imaging readout-based strategy is applied to quantify acrylamide. The developed aptasensor is novel for the naked-eye monitoring of the target through the color change of the solution inside the micropipette tip. The colorimetric aptasensor detects acrylamide in the concentration range of 0.05–200 nmol L−1 and at the trace level of 0.038 nmol L−1 with the comparable selectivity. The aptasensor can successfully quantify acrylamide in the chips, coffee, and bread samples with recoveries range from 92 to 102 %. The designed aptasensor paves an efficient device for the portable, on-site, facile, and real-time target sensing, superior for food safety control.

Metal-organic framework-enabled surface state passivation integrating with single-nuclease-propelled multistage amplification for ultrasensitive lab-on-paper photoelectrochemical biosensing

The surface states and photoinduced corrosion in the photoelectrode can severely reduce the photocurrent signal and stability of photoelectrochemical (PEC) sensor, which imposes a great challenge on the improvement of analytical performance. Herein, metal–organic framework (MOF)-enabled surface passivation strategy is proposed to passivate surface states, suppress photocorrosion and facilitate interfacial charge transport. In the concrete, zeolitic imidazolate framework-8 (ZIF-8), which serves as the passivation layer, is in-situ assembled on the surface of paper-based ZnO nanorods (NRs) array to restrain the charge trapping at the surface states and keep ZnO NRs from photocorrosion, thereby substantially enhancing the photocurrent signal and durability. Moreover, the unique single-nuclease-propelled multistage amplification (SNMA) and triple-helix molecular switch (THMS)-conformation-transition-mediated signal quenching strategies are also designed to further improve the sensitivity. The SNMA process can convert target microRNA-141 into quadruple amount of useful output DNA strands (trigger DNA) in one duplex-specific nuclease (DSN) cleavage cycle assisted by succedent DSN cleavage of DNA duplexes, guaranteeing a higher target amplification efficiency. Subsequently, the THMS structure which anchors double amount of signal enhancers to the photoelectrode surface can be disassembled by the resulted trigger DNA, therefore achieving powerful signal amplification. Profiting from the MOF-enabled surface state passivation and elegantly designed signal amplification strategies, the ultrasensitive detection of microRNA-141 is realized with a liner range from 0.2 fM to 2 nM and detection limit of 72 aM, which provides a promising paradigm for developing high-performance lab-on-paper PEC biosensing platform.

A self-powered 3D DNA walking machine triggered by triple-helix molecular switch for microRNA imagining in living cells

Herein, a novel self-powered 3D DNA walking machine triggered by triple-helix molecular switch (THMS) has been proposed for microRNA-21 (miRNA-21) imaging in living cells. Impressively, the 3D DNA walking machine was constructed with swing arm and THMS co-functionalized gold nanoparticles, THMS could precisely customize as a multifunctional structure with variable configuration. Once the 3D DNA walking machine was delivered into living cells, THMS was promptly disassembled due to the formation of DNA duplex (miRNA-21/TAMRA-DNA2), thus the 3D DNA walking machine was immediately triggered on AuNPs. Meanwhile, FAM-DNA1 quickly self-hybridized into hairpin and exposed the Mg2+ ribonucleoside adenosine (rA) recognition site in the loop region. Sequentially, swing arm hybridized with hairpin FAM-DNA1 and cleaved it with the assistance of Mg2+. Finally, numerous FAM-labeled segments were released from AuNPs and further paired with the spare part of TAMRA-DNA2 in the miRNA-21/TAMRA-DNA2 duplex, resulting in the two fluorescent dyes approaching each other, which induced the efficient FRET from FAM to TAMRA, thereby turned the system into “ON” state for signal amplification. It is noteworthy that the ingenious design of the self-powered 3D DNA walking machine avoids the external addition of fuel DNA strands or protein enzymes, which enables the autonomous motion of the 3D DNA walking machine in complex cellular microenvironment, greatly simplifies the experimental steps and improves the efficiency of the walking machine. Moreover, this assay will provide a novel signal amplification strategy for evaluating intracellular miRNA levels.

Triple-Helix Molecular Switch Triggered Cleavage Effect of DNAzyme for Ultrasensitive Electrochemical Detection of Chloramphenicol.

The abuse of chloramphenicol (CAP) in animal-derived products leads to serious food safety problems, so the sensitive and accurate determination of CAP residues has great noteworthiness for public health. Herein, we present a novel electrochemical aptasensor that incorporates a poly(diallyldimethylammonium chloride) functionalized graphene/Ag@Au nanosheets (PDDA-Gr/Ag@Au NSs) composite modified electrode and a DNAzyme signal amplification effect triggered by a triple-helix molecular switch (THMS) for detecting CAP. The PDDA-Gr/Ag@Au NSs composite has the advantages of high surface area, great conductivity, and dispersibility and has successfully improved the electrochemical performance of the electrode. Specific interaction with CAP will cause the signal transduction probe (STP) to be released from the THMS. After that, the DNAzyme will be activated with the help of Pb2+ and remove the immobilized signal probe on the electrode surface. The signal change was recorded by square wave voltammetry (SWV) and led to an accurate quantification of CAP. With all these features, the proposed sensing strategy yielded a satisfactory analytical performance with linearity between 1 pM and 1 μM and a limit of detection of 18.6 fM. Furthermore, the aptasensor shows excellent specificity for CAP in the presence of other antibiotics and resists interference with other common metal ions. Importantly, the performance is not diminished when the constructed aptasensor is applied to measuring CAP in milk powder. This THMS-based method is easy to design, and alteration to different targets can be achieved by simply replacing the aptamer sequence in the THMS. Therefore, this method shows significant prospects as a flexible platform for accurate monitoring of antibiotic residues in foodstuffs.

Highly sensitive and efficient fluorescent sensing for Hg2+ detection based on triple-helix molecular switch and exonuclease III-assisted amplification

Here, a novel fluorescent sensing for simple, highly sensitive and efficient detection of Hg2+ was developed as joint result of triple-helix molecular switch (THMS) and exonuclease III (Exo III)-assisted signal amplification. In this study, the special structure of THMS was used to realize efficient fluorescence quenching and excellent signal unit transformation to complete the output of signal FAM. In the absence of Hg2+, hairpin probe (HP) containing thymine-rich (T-rich) ssDNA strand can induce the dissociation of the THMS, causing FAM far away from BHQ1 and increasing fluorescence intensity. Nevertheless, Hg2+ could bind to the thymine (T) base to form the dsDNA with T-Hg2+-T structure that stimulates Exo III to digest it from the blunt 3′-terminus to 5′-terminus, causing Hg2+ to be released from the dsDNA. The released Hg2+ could initiate the next cycling, allowing a large number of hairpin probes to be cleaved by Exo III to form ssDNA. These ssDNA could inhibit the switch dissociation of THMS, causing a dramatic decrease in the fluorescence signal. This allowed for the highly sensitive detection of Hg2+ at concentrations as low as 1.04 pM. In addition, the sensing showed a linear detection range of 0.01–50 nM and was used for the assay of Hg2+ in real samples of Xiangjiang river water and tap water. These results showed that the provided fluorescent sensing has a good application prospect in environmental and food monitoring.

Light-Activated Nanodevice for On-Demand Imaging of miRNA in Living Cells via Logic Assembly.

Low-abundance biomarker amplification detection systems have been widely used to detect miRNAs; however, "always active" systems are insufficient for high spatial and temporal control of miRNAs. Here, we constructed a light-activated nanodevice (LAN) based on DNA nanotechnology for high spatial and temporal precision detection of low-abundance miRNA. Light-activated hairpin probes and triple-helix molecular switches were modified on the surface of gold nanoparticles (AuNPs) to trigger miRNA on-demand imaging analysis by UV light activation. In the presence of both UV light and miRNA, the LAN releases hairpin DNA and completes the hybridization chain reaction (HCR) with the conformation-altered triple-helix molecular switch, enabling fluorescence imaging of low-abundance miRNAs in living cells. The current work provides an opportunity to develop light-activated signal amplification sensors that can accurately image miRNAs on-demand in both temporal and spatial dimensions.

A Self-Powered 3d DNA Walking Machine Triggered by Triple-Helix Molecular Switch for Microrna Imagining in Living Cells

A Self-Powered 3d DNA Walking Machine Triggered by Triple-Helix Molecular Switch for Microrna Imagining in Living Cells

A Self-Powered 3d DNA Walking Machine Triggered by Triple-Helix Molecular Switch for Microrna Imagining in Living Cells

A Self-Powered 3d DNA Walking Machine Triggered by Triple-Helix Molecular Switch for Microrna Imagining in Living Cells

A versatile signal-on electrochemical biosensor for Staphylococcus aureus based on triple-helix molecular switch

An electrochemical biosensor for Staphylococcus aureus (S. aureus) is designed based on a triple-helix molecular switch which can control the switching of electrochemical signals. When S. aureus exists in the solution, the aptamer binds to the target and releases the complementary sequence (cDNA) from the magnetic beads to the solution. With the help of the primer, the strand displacement amplification (SDA) reaction carries out under the action of Bsm DNA polymerase and Nb.bpu10I endonuclease, which produces numerous ssDNA probe. Subsequently, the interaction between ssDNA probe and the molecular switch (MS) leads to the dissociation of the triple-helix DNA structure on the electrode, which further triggers the formation of a large number of electroactive G-quadruplex/hemin complex, and turns on the electrochemical signal. The biosensor showed a broad dynamic range from 30 to 3 × 108 CFU/mL, with a low detection limit of 8 CFU/mL. In addition, the sensor is used for the detection of S. aureus in lake water, tap water and honey samples, indicating that the sensor has good practicability. Due to the special design of cDNA and primer sequences, the same principle has been successfully applied in configuration of biosensor for Escherichia coli (E. coli). In summary, the constructed sensor has the advantages of high sensitivity, high specificity and versatility, which provides a promising alternative method for the detection of various harmful bacteria in food and environment, and has a certain application prospect.

An ultrasensitive platform for PCB77 detection: New strategy for liquid crystal-based aptasensor fabrication

Polychlorinated biphenyls (PCBs) are considered persistent bio-accumulative toxicants which threats global food safety and environmental health. Traditional analytical techniques for detection of PCBs are time-consuming and they do not satisfy urgent need for rapid and accurate monitoring of these persistent pollutants. Biosensor technology may be promising in this respect. Here we demonstrate a novel liquid crystal (LC)-based aptasensing platform as a promising label-free and rapid biosensor for PCB77 detection. This novel molecular strategy utilize triple-helix molecular conformational switch which is mediated formation of duplex on sensing platform in presence of target. Duplex forming leads to optical change from dark to bright in a liquid crystal based aptasensor. The limit of quantification of the LC-aptasensor to PCB77 is 1.5 × 10−5 μg/L with comparable selectivity. Besides, we also demonstrated that this system is able to detect PCB77 in tap water, environmental water and milk. This strategy has potential for label-free and portable detection of different targets without any aptamer sequence length restrictions.

A PNA-DNA2 Triple-Helix Molecular Switch-Based Colorimetric Sensor for Sensitive and Specific Detection of microRNAs from Cancer Cells.

Peptide nucleic acids (PNAs), the synthetic DNA mimics that can bind to oligonucleotides to form duplexes, triplexes, and quadruplexes, could be advantageous as probes for nucleic acid sequences owing to their unique physicochemical and biochemical properties. We have found that a homopurine PNA strand could bind to two homopyrimidine DNA strands to form a PNA-DNA2 triplex. Moreover, the cyanine dye DiSC2 (5) could bind with high affinity to this triplex and cause a noticeable color change. On the basis of this phenomenon, we have designed a label-free colorimetric sensing platform for miRNAs from cancer cells by using a PNA-DNA2 triple-helix molecular switch (THMS) and DiSC2 (5). This sensing platform can detect miRNA-21 specifically with a detection limit of 0.18 nM, which is comparable to that of the THMS-mediated fluorescence sensing platform. Moreover, this colorimetric platform does not involve any chemical modification or enzymatic signal amplification, which boosts its applicability and availability at the point of care in resource-limited settings. The universality of this approach can be simply achieved by altering the sequences of the probe DNA for specific targets.

A label-free resonance light scattering biosensor for nucleic acids using triple-helix molecular switch and G-quadruplex nanowires

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.

Establishment of a universal and sensitive plasmonic biosensor platform based on the hybridization chain reaction (HCR) amplification induced by a triple-helix molecular switch

Herein, we established a universal and sensitive plasmonic sensing strategy for biomolecule assays by coupling the hybridization chain reaction (HCR) strategy and a triple-helix molecular switch. Upon the recognition of the target, a single-stranded DNA as a universal trigger (UT) was released from the triple-helix molecular switch (THMS). Thus, the HCR process can be triggered between two hairpins M1 and M2, resulting in the aggregation of gold nanoparticles (AuNPs) via the hybridization between the tail sequence on M1 (or M2) and a DNA-AuNP probe with a dramatic change in the absorbance at 521 nm. More specifically, the strategy, which was conducted by the introduction of target-specific recognition of THMS and universalized by virtue of altering the aptamer or DNA sequence without changing the triple-helix structure, enables simple design for multiple target detection. By taking advantage of THMS, this strategy could enable stable and sensitive detection of a variety of targets including nucleic acids, small molecules and proteins, which may possess great potential for practical applications.

A triple-helix molecular switch photoelectrochemical biosensor for ultrasensitive microRNA detection based on position-controllable CdS//CdTe signal enhancement and switching.

Herein, a triple-helix molecular switch photoelectrochemical (PEC) biosensor is developed for ultrasensitive and selective detection of microRNA based on position-controllable CdS//CdTe signal enhancement and switching accompanying the signal amplification of a three-dimensional DNA walking machine. The developed PEC biosensor exhibits excellent analytical performance for microRNA-141 detection with a wide linear range from 5 aM to 100 fM, a low detection limit of 1.3 aM and outstanding selectivity.