Triple-helix molecular-switch-actuated rolling circle amplification and catalytic hairpin assembly multistage signal amplified fluorescent aptasensor for detection of aflatoxin B1.

BACKGROUND: Mycotoxins, a class of secondary metabolites produced by molds, are widely distributed in nature and are very common in food contamination. Aflatoxin B1 (AFB1) is a highly stable natural mycotoxin, and many agricultural products are easily contaminated by AFB1, it is important to establish a sensitive and efficient AFB1 detection method for food safety. The fluorescence aptamer sensor has shown satisfactory performance in AFB1 detection, but most of the fluorescence aptasensors are not sensitive enough, so improving the sensitivity of the aptasensor becomes the focus of this work. RESULTS: Herein, an innovative fluorescent aptasensor for AFB1 detection which is based on catalytic hairpin assembly (CHA) and rolling circle amplification (RCA) driven by triple helix molecular switch (THMS) is proposed. A functional single-strand with an AFB1 aptamer, here called an APF, is first designed to lock onto the signal transduction probe (STP), which separates from THMS when target AFB1 is present. Subsequently, STP initiates the RCA reaction along the circular probe, syntheses macro-molecular mass products through repeated triggering sequences, triggers the CHA reaction to produce a large number of H1-H2 structures, which causes FAM to move away from BHQ-1 and recover its fluorescence signal. The fluorescence signal from FAM at 520 nm was collected as the signal output of aptasensor in this work. With high amplification efficiency of RCA and CHA of the fluorescence sensor, resulting in a low LOD value of 2.95 pg mL-1(S/N = 3). SIGNIFICANCE: The successful establishment of the sensor designed in this work shows that the cascade amplification reaction is perfectly applied in the fluorescent aptamer sensor, and the signal amplification through the reaction between DNA strands is a simple and efficient method. In addition, it's also important to remember that the aptasensor can detect other targets only by changing the sequence of the aptamer, without redesigning other DNA sequences in the reaction system.

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.

Accurate and rapid quantification of PD-L1 positive exosomes by a triple-helix molecular probe.

Programmed death ligand-1 (PD-L1) positive exosomes (P-Exo) have been widely used for tumor diagnosis. However, accurate and rapid quantification of P-Exo remains challenging due to the heterogeneity of clinical individuals and isolation techniques. In this study, the triple-helix molecular probe (THMP) coupled with high-affinity silica-based TiO2 magnetic beads was used to isolate exosomes and to analyze the relative abundance of P-Exo in total exosomes (T-Exo). By employing this strategy, the entire analysis was completed within 70 min and the detection limit for P-Exo was 880 particles µL-1. Additionally, the relative abundance of P-Exo in T-Exo (RAP-Exo/T-Exo) was calculated from their fluorescence ratio, which could avoid errors due to differences in samples and separation methods, and identify 1.5 × 103 P-Exo from 5 × 106 T-Exo per microliter. RAP-Exo/T-Exo values were not only effective in distinguishing healthy volunteers from breast cancer patients, but also highly positively correlated with the stage of breast carcinoma. Overall, this strategy opens a new avenue for rapid and quantitative analysis of P-Exo, providing an opportunity for precise diagnosis and prediction of treatment efficacy in cancer.

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.

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 simple fluorescent strategy based on triple-helix molecular switch for sensitive detection of chloramphenicol.

A simple fluorescent strategy based on the formation of triple-helix molecular switch (THMS) between a signal transduction probe (STP) and an aptamer (Apt) was constructed for the determination of chloramphenicol (CAP). A weak fluorescence intensity was observed for STP solution due to the proximity of fluorophore and quencher through intramolecular DNA hybridization, causing the fluorescence quenching. The fluorescence intensity of the system was significantly enhanced after the addition of Apt. It was attributed to the formation of THMS between the Apt and STP through the Watson-Crick and Hoogsteen base pairing, resulting in the restoration of fluorescence because of the long distance between the fluorophore and quencher of STP. The fluorescence intensity of the system decreased due to the release of STP caused by the specific binding between Apt and CAP. The quantitative analysis of CAP could be achieved based on the decreased fluorescence intensity. The parameters affecting the performance of THMS including the Apt arm length, pH of buffer solution, Mg2+ concentration and the formation time of THMS were investigated in detail. Under the optimal conditions (Apt arm length of 9 bases, pH of 6.5, 2.5 × 103 µmol L-1 Mg2+, THMS formation time of 30 min), the decreased fluorescence intensity and the concentration of chloramphenicol were linear in the range of 5.0 × 10-3-2.0 × 10-1 µmol L-1 with the correlation coefficient of 0.9963. The limit of detection was 1.2 nmol L-1. Subsequently, the developed method was applied to the analysis of chloramphenicol in honey sample, and the recovery was between 84.5% and 103.0% with relative standard deviation less than 4.6%.

Signal-switchable lab-on-paper photoelectrochemical aptasensing system integrated triple-helix molecular switch with charge separation and recombination regime of type-II CdTe@CdSe core-shell quantum dots.

Herein, a new "on-off-on" signal switch system combined triple helix molecular switch with efficient charge separation and transfer between different sensitization units was designed for the ultrasensitive photoelectrochemical (PEC) determination of prostate-specific antigen (PSA). Concretely, the initial "signal-on" state was obtained via the cascaded sensitization structure consisting of type-II CdTe@CdSe core-shell quantum dots (QDs), CdS QDs, and ZnO nanotubes, which were assembled on Au nanoparticles modified paper fibers with the aid of signal transduction probe (STP). Thereinto, the type-II CdTe@CdSe QDs with hole-localizing core and electron-localizing shell could enable the ultrafast charge transfer and retard the charge recombination, magnifying the initial photocurrent response and preserving the high efficiency of signal-switchable PEC aptasensing system. Subsequently, the PSA aptamer (PSA-Apt) modified with gold nanoparticles (GNPs) was introduced by the hybridization of PSA-Apt with STP and the hairpin configuration of STP changed from closed to open state, forming a triple-helix structure. Hence, the CdTe@CdSe QDs labeled on the terminal of STP moved away from the electrode surface while the GNPs kept attached close to it. The proposed aptasensor turned to "signal-off" state because of the dual inhibition of vanished cosensitization effect and signal quenching effect of GNPs. Upon the target recognition, the triple-helix structure was perturbed with the formation of DNA-protein complex and the recovery of STP hairpin structure, resulting in the second "switch-on" state. Based on the target-induced photocurrent enhancement, the proposed PEC aptasensor was utilized for the determination of PSA with high sensitivity, persuasive selectivity, and excellent stability.

Photoelectrochemical biosensor of HIV-1 based on cascaded photoactive materials and triple-helix molecular switch.

In this work, an ultrasensitive photoelectrochemical (PEC) biosensor was proposed to detect nucleic acids on the basis of cascaded photoactive materials and triple-helix molecular switch. DNA sequence of human immunodeficiency virus type 1 (HIV-1) was chosen as the target DNA (T-DNA). Cascaded photoactive structure was formed via different sizes of CdTe quantum dots (QDs) sensitized ZnO nanorods (ZnO NRs), which was employed as a cascaded photoactive interface to amplify the photocurrent signal. A hairpin structure DNA (H-DNA) as capture probe was conjugated onto the photoactive interface through amide bond, and then a single-stranded DNA modified with gold nanoparticles labeled alkaline phosphatase (ALP-Au NPs-DNA) at each end was introduced to hybridize with the H-DNA to form a triple-helix conformation. The T-DNA detection was based on the photocurrent response change resulted from conformation change of the triple-helix molecule after hybridization with T-DNA. In the absence of T-DNA, the triple-helix molecule was in a closed state and the ALP of ALP-Au NPs-DNA could specifically catalyze the ascorbic acid 2-phosphate (AAP) to generate ascorbic acid (AA) as electron donors, which resulted in a significant photocurrent response due to the rapid electron transfer process. However, in the presence of T-DNA, the T-DNA hybridized with the ALP-Au NPs-DNA molecule, which caused triple-helix molecule in an opened state and compelled ALP-Au NPs-DNA away from the electrode surface, resulting in the absence of ALP which could catalyze AAP to generate AA. Subsequently, the photocurrent response significantly decreased. The proposed PEC biosensor not only had a wide detection range of 1fM-1nM and low detection limit (0.65 fM), but also showed excellent reproducibility, specificity and stability, which had great application prospect and opened up a new research method in the early clinical diagnosis and cancer research.

Ultrasensitive aptamer biosensor for arsenic (III) detection based on label-free triple-helix molecular switch and fluorescence sensing platform.

Arsenic ion is a well-known harmful heavy element widely existing in the environment. Arsenic pollution occurring frequently has become increasing a serious worldwide threat to human health and the environment. The development of sensitive and reliable methods to detect As3+ in water is of great importance to biochemical research and monitoring applications. Herein, a label-free fluorescence sensing platform was elaborately designed for As3+ monitoring using exonuclease III (Exo III)-assisted cascade target recycling amplification strategy. The triple-helix molecular switch was employed as the sensing element and 2-amino-5,6,7-trimethyl-1,8-naphthyridine was used as the signal indicator. The resulting biosensor is simple, ultrasensitive, and exhibits a limit of detection of 5 ng/L with high selectivity. Meanwhile, the proposed sensor is successfully applied to determination of As3+ in practical sample analysis (tap water, lake water and pond water). The results shown herein have important implications in the development of new fluorescent sensors for the fast, easy, and selective detection and quantification of As3+ in water samples. More importantly, the proposed platform can be extended to detect other heavy metal ions with newly designed triple-helix molecular switch, as well as pesticide residue, antibiotic residues, and biomarkers by using aptamer sequences.

Triple-helix molecular switch-based aptasensors and DNA sensors.

Utilization of traditional analytical techniques is limited because they are generally time-consuming and require high consumption of reagents, complicated sample preparation and expensive equipment. Therefore, it is of great interest to achieve sensitive, rapid and simple detection methods. It is believed that nucleic acids assays, especially aptamers, are very important in modern life sciences for target detection and biological analysis. Aptamers and DNA-based sensors have been widely used for the design of various sensors owing to their unique features. In recent years, triple-helix molecular switch (THMS)-based aptasensors and DNA sensors have been broadly utilized for the detection and analysis of different targets. The THMS relies on the formation of DNA triplex via Watson-Crick and Hoogsteen base pairings under optimal conditions. This review focuses on recent progresses in the development and applications of electrochemical, colorimetric, fluorescence and SERS aptasensors and DNA sensors, which are based on THMS. Also, the advantages and drawbacks of these methods are discussed.

Amplified Fluorescent Detection of Mercuric Ions by Conjugation of the ThT-induced G-Quadruplex Based Hybridization Chain Reaction.

A sensitive fluorescent method for the detection of Hg2+ was developed based on triple-helix molecular switch (THMS)-induced hybridization chain reaction (HCR) amplification. THMS was composed of a T-rich mercury-specific probe and an initiator probe, designed by the Watson-Crick and Hoogsteen base pairings and employed as a signal trigger. Two hairpin probes containing the G-quadruplex sequence were used as signal amplification elements. In the presence of Hg2+, the T-Hg2+-T mismatch resulted in disassembling the THMS and releasing the initiator probe. One of the hairpins was opened by the released initiator probe, which triggered a successive cross-opening of two hairpins based the strand displacement principle, resulting in the formation of long-chain DNA with multiple G-quadruplex. When thioflavin T (ThT), a fluorophore, was bound to the G-quadruplex, an obvious fluorescence enhancement would occur. This sensing system enabled the highly sensitive and selective detection of aqueous Hg2+ with a limit-of-detection of 10.2 pM.

Aptamer contained triple-helix molecular switch for rapid fluorescent sensing of acetamiprid.

In this study, an aptamer-based fluorescent sensing platform using triple-helix molecular switch (THMS) was developed for the pesticide screening represented by acetamiprid. The THMS was composed of two tailored DNA probes: a label-free central target specific aptamer sequence flanked by two arm segments acting as a recognition probe; a hairpin-shaped structure oligonucleotide serving as a signal transduction probe (STP), labeled with a fluorophore and a quencher at the 3' and 5'-end, respectively. In the absence of acetamiprid, complementary bindings of two arm segments of the aptamers with the loop sequence of STP enforce the formation of THMS with the "open" configuration of STP, and the fluorescence of THMS is on. In the presence of target acetamiprid, the aptamer-target binding results in the formation of a structured aptamer/target complex, which disassembles the THMS and releases the STP. The free STP is folded to a stem loop structure, and the fluorescence is quenched. The quenched fluorescence intensity was proportional to the concentration of acetamiprid in the range from 100 to 1200nM, with the limit of detection (LOD) as low as 9.12nM. In addition, this THMS-based method has been successfully used to test and quantify acetamiprid in Chinese cabbage with satisfactory recoveries, and the results were in full agreement with those from LC-MS. The aptamer-based THMS presents distinct advantages, including high stability, remarkable sensitivity, and preservation of the affinity and specificity of the original aptamer. Most importantly, this strategy is convenient and generalizable by virtue of altering the aptamer sequence without changing the triple-helix structure. So, it is expected that this aptamer-based fluorescent assay could be extensively applied in the field of food safety inspection.

A novel colorimetric triple-helix molecular switch aptasensor for ultrasensitive detection of tetracycline.

Detection methods of antibiotic residues in blood serum and animal derived foods are of great interest. In this study a colorimetric aptasensor was designed for sensitive, selective and fast detection of tetracycline based on triple-helix molecular switch (THMS) and gold nanoparticles (AuNPs). As a biosensor, THMS shows distinct advantages including high stability, sensitivity and preserving the selectivity and affinity of the original aptamer. In the absence of tetracycline, THMS is stable, leading to the aggregation of AuNPs by salt and an obvious color change from red to blue. In the presence of tetracycline, aptamer binds to its target, signal transduction probe (STP) leaves the THMS and adsorbs on the surface of AuNPs. So the well-dispersed AuNPs remain stable against salt-induced aggregation with a red color. The presented aptasensor showed high selectivity toward tetracyclines with a limit of detection as low as 266 pM for tetracycline. The designed aptasensor was successfully applied to detect tetracycline in serum and milk.