Substrate Strand Research Articles

Microfluidic bead-based biosensor: Ultrasensitive ctDNA detection based on duplex-functional split-DNAzyme and dendritic enzyme-free signal amplification

Circulating tumor DNA (ctDNA) is a crucial cancer biomarker for early or noninvasive monitoring, which is essential for developing ultrasensitive and selective assays in cancer diagnosis and treatment. Herein, a cascade signal amplification of duplex-functional split-DNAzyme and dendritic probes was proposed for ultrasensitive and specific detection of nasopharyngeal carcinoma-associated Epstein-Barr virus (EBV) DNA on microfluidic microbead array chips. With the assistance of Pb2+, the duplex-functional split-DNAzyme recognizes EBV DNA and then rapidly cleaves the substrate strand. Subsequently, the released target could be recycled, and its exposed capture probe, triggered the dendritic enzyme-free signal amplification. As the enhanced mass transfer capability, target recycling, and dendritic DNA structure signal amplification inherent to microfluidic bead arrays were integrated, it achieved an excellent detection limit of 0.36 fM and a wide linear range of 1 fM∼103 fM. Further, it was applied to content detect simulated samples of EBV DNA, recovery ranged from 97.2 % to 108.1 %, and relative standard deviation (RSD) from 3.3 % to 5.9 %, exhibiting satisfactory recovery results. The developed microfluidic biosensor was a high-sensitivity and anti-interference system for ctDNA analysis, with minimal reagent volumes (microlitres) required. Thus, it is a promising platform for ctDNA at the lowest level at their earliest incidence.

A colorimetric and fluorescent dual-mode sensor based on bifunctional G-quadruplex-hemin complex for the determination of Pb2+

Due to the threat of lead pollution to health, environmental and food safety, developing simple and fast detection methods is highly required. Whereas, traditional single-mode probe suffers from limited application scenario. In this study, a colorimetric and fluorometric dual-mode probe for Pb2+ determination was constructed by using bifunctional G-quadruplex-hemin complex. In this dual-mode probe, enzyme strand and substrate strand of 8–17 DNAzyme are labeled with G-quadruplex-hemin complex and fluorophore, respectively. In the absence of Pb2+, the self-assembly of enzyme strand and substrate strand inhibits intrinsic mimic peroxidase of G-quadruplex-hemin complex by base-pairing, which also quench the fluorescence via in proximity effect. When the DNAzyme is activated by Pb2+, the quenched fluorescence is restored as well as the inherent peroxidase mimetic activity, leading to dual signal output. Under optimal conditions, this dual-mode probe exhibit a good linear relationship between logarithm of Pb2+ concentration and signal difference within the range from 1.5 nM to 20 nM and 0.5 nM to 10 nM for colorimetric and fluorescence mode, respectively. The detection limits for the corresponding mode were estimated to be 1.29 nM and 0.16 nM, respectively. This dual-mode probe also successfully applied for the spiked river water assay with satisfactory recovery in the range of 93.2 %–115.3 %. This work paves a new way for DNAzyme based dual-mode probe construction.

Spatiotemporal control of DNAzyme activity for fluorescent imaging of telomerase RNA in living cells

BackgroundHuman telomerase is a ribonucleoprotein complex that includes proteins and human telomerase RNA (hTR). Emerging evidence suggested that the expression level of hTR was high related with the development of tumor, so it is important to accurately detect the content of hTR. Optical control of DNAzyme activity shows a promising strategy for precise biosensing, biomedical imaging and modulation of biological processes. Although DNAzyme-based sensors can be controlled spatiotemporally by light, its application in the detection of hTR in living cells is still rare. Therefore, designing DNAzyme activity spatiotemporal controllable sensors for hTR detection is highly needed. ResultsWe developed a UV light-activated DNAzyme-based nanoprobe for spatially accurate imaging of intracellular hTR. The proposed nanoprobe was named MDPH, which composed of an 8–17 DNAzyme (D) inactivated by a protector strand (P), a substrate strand (H), and MnO2 nanosheets. The MnO2 nanosheets can enhance the cellular uptake of DNA strands, so that MDPH probe can enter cells autonomously through endocytosis. Under the high concentration of GSH in cancer cells, MnO2 nanosheets can self-generate cofactors to maintain the catalytic activity of DNAzyme. When exposing UV light and in presence of target hTR, DNAzyme could cleave substrate H, resulting in the recovery of fluorescence of the system. The cells imaging results show that MDPH probe could be spatiotemporally controlled to image endogenous hTR in cancer cells. SignificanceWith this design, telomerase RNA-specific fluorescent imaging was achieved by MDPH probe in both cancer and normal cells. Our probe made a promising new platform for spatiotemporal controllable intracellular hTR monitoring. This current method can be applied to monitor a variety of other biomarkers in living cells and perform medical diagnosis, so it may has broad applications in the field of medicine.

Fluorescence biosensor for ultrasensitive detection of the available lead based on target biorecognition-induced DNA cyclic assembly

A fluorescence biosensor was developed for the ultrasensitive detection of the available lead in soil samples by coupling with DNAzyme and hairpin DNA cyclic assembly. The biorecognition between lead and 8-17 DNAzyme will cleave the substrate strands (DNA2) and release the trigger DNA (T), which can be used to initiate the DNA assembly reactions among the hairpins (H1, H2, and H3). The formed Y-shaped sensing scaffold (H1-H2-H3) contains active Mg2+-DNAyzmes at three directions. In the presence of Mg2+, the BHQ and FAM modified H4 will be cleaved by the Mg2+-DNAyzme to generate a high fluorescence signal for lead monitoring. The linear range of the fluorescence biosensor is from 1 pM to 100 nM and the detection limit is 0.2 pM. The biosensor also exhibited high selectivity and the nontarget competing heavy metals did not interfere with the detection results. Compare with the traditional method (DTPA+ICP-MS) for the available lead detection, the relative error (Re) is in the range from −8.3 % to 9.5 %. The results indicated that our constructed fluorescence biosensor is robust, accurate, and reliable, and can be applied directly to the detection of the available lead in soil samples without complex extraction steps.

A DNAzyme dual-feedback autocatalytic exponential amplification biocircuit for microRNA imaging in living cells

Autocatalytic biocircuit are powerful tools for analysing intracellular biomarkers, but these tools are constrained by limitations in amplification capacity and intracellular delivery efficiency. In this work, we developed a DNAzyme-based dual-feedback autocatalytic exponential amplification biocircuit sustained by a honeycomb MnO2 nanosponge (EDA2@hMNS) for live-cell imaging of intracellular low-abundance microRNAs (miRNA). The EDA2 biocircuit comprises a blocked DNAzyme (b-DNAzyme), a Fuel strand and a Substrate strand. In the EDA2 biocircuit, target miRNAs are recycled and feedback for rounds of DNAzymatic amplification, and the DNAzymatic reactions continuously generate target miRNA analogues for dual-feedback to achieve multiple parallel cascade DNAzymatic reactions that improve amplification capacity substantially. In addition, the hMNS ensures high loading and delivery efficiency of biocircuit probes into living cells and also provides sufficient Mn2+ DNAzyme cofactor from in situ decomposition by intracellular glutathione (GSH). The EDA2@hMNS realized a detection limit of 17 pM, which is 288-fold lower than the b-DNAzyme lacking the DNAzymatic amplification. These results demonstrate the great promise for this critical tool in analysing low-abundance biomarkers and cancer diagnostics.

Ratiometric Detection of Zn2+ Using DNAzyme-Based Bioluminescence Resonance Energy Transfer Sensors.

While fluorescent sensors have been developed for monitoring metal ions in health and diseases, they are limited by the requirement of an excitation light source that can lead to photobleaching and a high autofluorescence background. To address these issues, bioluminescence resonance energy transfer (BRET)-based protein or small molecule sensors have been developed; however, most of them are not highly selective nor generalizable to different metal ions. Taking advantage of the high selectivity and generalizability of DNAzymes, we report herein DNAzyme-based ratiometric sensors for Zn2+ based on BRET. The 8-17 DNAzyme was labeled with luciferase and Cy3. The proximity between luciferase and Cy3 permiQed BRET when coelenterazine, the substrate for luciferase, was introduced. Adding samples containing Zn2+ resulted in a cleavage of the substrate strand, causing dehybridization of the DNAzyme construct, thus increasing the distance between Cy3 and luciferase and changing the BRET signals. Using these sensors, we detected Zn2+ in serum samples and achieved Zn2+ detection with a smartphone camera. Moreover, since the BRET pair is not the component that determines the selectivity of the sensors, this sensing platform has the potential to be adapted for the detection of other metal ions with other metal-dependent DNAzymes.

A turn-on fluorescence strategy for hypochlorous acid detection based on DNAzyme-assisted cyclic signal amplification

Hypochlorous acid (HClO) is a crucial active oxygen component and one of the innate immunity's barrier substances in the body. Abnormal fluctuations in HClO concentration can lead to increased oxidative stress, cellular dysfunction, and the onset of various diseases. Thus, developing convenient, affordable, efficient, and sensitive methods to monitor HClO concentration in healthcare and pathophysiology research is highly significant. In this study, we developed a novel fluorescence strategy for HClO detection based on nucleic acid oxidative cleavage and Pb2+-dependent DNAzyme. By introducing a phosphorothioate site in the hairpin-structured nucleic acid sequence, the nucleic acid probe specifically recognized HClO and underwent oxidative cleavage. Upon cleavage, the enzyme strand is liberated, forming DNAzyme. This DNAzyme then cleaves the substrate strand, liberating the initially quenched fluorescent dyes and generating a turn-on fluorescent signal. The enzyme strand produced by the oxidative cleavage of HClO can be repeatedly utilized, thus realizing the cyclic signal amplification to reduce background noise. We verified the detection mechanism of this strategy through stepwise fluorescence spectroscopy analysis and electrophoresis. Under optimal experimental conditions, the method achieved a detection limit of 5.38 nM and a linear range of 1 nM-800 nM. This method demonstrated exceptional performance in actual biological sample testing and presented an exciting opportunity for expanded utilization in clinical diagnosis and medical research.

3D DNAzyme walker based electrochemical biosensor for attomolar level microRNA-155 detection

Herein, an ultrasensitive electrochemical biosensor for microRNA-155 (miR-155) detection based on the powerful catalytic and continuous walking signal amplification capability of 3D DNAzyme walker and the gold nanoparticles/graphene aerogels carbon fiber paper-based (AuNPs/GAs/CFP) flexible sensing electrode with excellent electrochemical performance was successfully constructed. In a proof-of-concept experiment, in the presence of miR-155, the DNAzyme strands anchored on the streptavidin-modified magnetic beads (MBs) silenced by locked strands can be activated, thus generating the walking arm of the 3D DNAzyme walker. Meanwhile, the substrate strands modified with Fe-MOF-NH2 nanoparticles were evenly distributed on the surface of MBs and served as tracks of the 3D DNAzyme walker. Once the DNAzyme strand was activated, the catalytic site in the substrate strand can be cleaved in the presence of Mn2+, and a large number of stumps modified with Fe-MOF-NH2 nanoparticles (output@Fe-MOF-NH2) will be generated during the continuous and efficient walking cleavage of the DNAzyme walker, driving the recognition-catalysis-release cycle process for signal amplification. Immediately afterwards, the signal was read out through the base complementary pairing of capture probe (PS) immobilized on the surface of the paper-based flexible sensing electrode AuNPs/GAs/CFP and signal probes output@Fe-MOF-NH2, thus achieving the quantitative detection of miR-155. Under optimal experimental conditions, the designed 3D DNAzyme walker-based biosensor exhibited a relatively lower limit of detection (LOD) of 56.23 aM, with a linear range of 100 aM to 100 nM. Overall, the proposed 3D DNAzyme walker biosensor exhibited good interference and reproducibility, demonstrating a promising future in the field of clinical disease diagnosis.

Dual-Responsive Turn-On T1 Imaging-Guided Mild Photothermia for Precise Apoptotic Cancer Therapy.

Apoptosis has gained increasing attention in cancer therapy as an intrinsic signaling pathway, which leads to minimal leakage of waste products from a dying cell to neighboring normal cells. Among various stimuli to trigger apoptosis, mild hyperthermia is attractive but confronts limitations of non-specific heating and acquired resistance from elevated expression of heat shock proteins. Here, a dual-stimulation activated turn-on T1 imaging-based nanoparticulate system (DAS) is developed for mild photothermia (≈43°C)-mediated precise apoptotic cancer therapy. In the DAS, a superparamagnetic quencher (ferroferric oxide nanoparticles, Fe3 O4 NPs) and a paramagnetic enhancer (Gd-DOTA complexes) are connected via the N6-methyladenine (m6 A)-caged, Zn2+ -dependent DNAzyme molecular device. The substrate strand of the DNAzyme contains one segment of Gd-DOTA complex-labeled sequence and another one of HSP70 antisense oligonucleotide. When the DAS is taken up by cancer cells, overexpressed fat mass and obesity-associated protein (FTO) specifically demethylates the m6 A group, thereby activating DNAzymes to cleave the substrate strand and simultaneously releasing Gd-DOTA complex-labeled oligonucleotides. The restored T1 signal from the liberated Gd-DOTA complexes lights up the tumor to guide the location and time of deploying 808nm laser irradiation. Afterward, locally generated mild photothermia works in concert with HSP70 antisense oligonucleotides to promote apoptosis of tumor cells. This highly integrated design provides an alternative strategy for mild hyperthermia-mediated precise apoptotic cancer therapy.

A ratiometric fluorescence platform for lead ion detection via RNA cleavage-inhibited self-assembly of three-arm branched junction

Heavy metal pollution can pose a threat to food safety and human health, and accurate quantification of heavy metal ions is a vital requirement. Emerging DNA nanostructures-based biosensors offer attractive tools toward ultra-sensitive or rapid analysis of heavy metal ions. However, the problems including complex design, severe reaction conditions and undesirable reliability are inevitable obstacle in advancing their extension and application. Herein, a ratiometric fluorescent platform was established for monitoring lead ion (Pb2+) in food based on dual Förster resonance energy transfer (FRET) and RNA cleavage-inhibited self-assembly of three-arm branched junction (TBJ). GR-5 DNAzyme was employed for Pb2+ recognition, and enzyme-free amplification technique catalytic hairpin assembly (CHA) served to form FRET probes-carried TBJ. The substrate strand (S) of DNAzyme triggered the generation of CHA-TBJ, and Pb2+-responsive cleavage of S hindered the assembly of CHA-TBJ, causing opposite changes in the FRET states of FAM/BHQ1 and ROX/BHQ2 pairs. The fluorescence responses were recorded through synchronous fluorescence spectrometry to indicate Pb2+ concentration, allowing sensitive and reliable identification of Pb2+ in the linear range of 0.05–5 ng mL−1 with the detection limit of 0.03 ng mL−1. The Pb2+ detection can be achieved under conventional reaction conditions, simple mixing procedures and one-step measurement operation. The approach can afford excellent specificity for Pb2+ against competing metal ions, and can be applied to analyze Pb2+ in tea samples with satisfactory results. This facile fluorescence platform shows a capable method for Pb2+ detection, and provides new avenue in the development of ratiometric approaches and DNAzyme strategies for monitoring heavy metal pollution, facilitating the transformation of DNAzyme-based biosensors for food safety control.

Self-Powered DNAzyme Walker Enables Dual-Mode Biosensor Construction for Electrochemiluminescence and Electrochemical Detection of MicroRNA.

Herein, an electrochemiluminescence (ECL) and electrochemical (EC) dual-mode biosensor platform with a self-powered DNAzyme walking machine was established for accurate and sensitive detection of miRNA-21. By employing a magnesium ion (Mn2+)-dependent DNAzyme cleavage cycling reaction, the walking machine was built by assembling DNAzyme walking strands and ferrocene (Fc)-labeled substrate strands on the Au nanoparticles and graphitic carbon nitride nanosheet (g-C3N4 NS)-covered electrode. The DNAzyme walking strand was first prohibited by a blocker strand. After the addition of target miRNA-21 and Mn2+, the DNAzyme walker could be activated and produce autonomous movements along the electrode track fueled by Mn2+-dependent DNAzyme-catalyzed substrate cleavage without additional energy supply. Notably, each walking step resulted in the cleavage of a substrate strand and the release of a Fc-labeled DNA strand fragment, allowing us to acquire an extreme ECL signal recovery of g-C3N4 inhibited by Fc. Meanwhile, numerous Fc-labeled DNA fragments escaped from the surface of the electrode, directly producing an obvious decrease in the square wave voltammetry (SWV) signal from Fc on the same sensing platform. This work not only avoided difficultly assembling various signal indicators but also significantly improved the sensitivity through using self-powered DNAzyme-walker amplification. Moreover, the proposed design employed the same reaction to produce two signal output modes, which could eliminate the interference from diverse reactive pathways on the outcome to mutually improve the accuracy. Therefore, the dual-mode miRNA-21 biosensor exhibited wide detection ranges of 100 aM to 100 nM with low detection limits of 54.3 and 78.6 aM by ECL and SWV modes, respectively, which provided an efficient and universal biosensing approach with extensive applications in early disease diagnosis and bioanalysis.

A Novel Cu2+ Quantitative Detection Nucleic Acid Biosensors Based on DNAzyme and “Blocker” Beacon

In this paper, a "turn-off" biosensor for detecting copper (II) ions based on Cu2+-dependent DNAzyme and a "blocker" beacon were developed. Upon the copper ion being added, the Cu2+-dependent DNAzyme substrate strand was irreversibly cleaved, thereby blocking the occurrence of the ligation reaction and PCR, which inhibited the G-rich sequence from forming the G-quadruplex structure, efficiently reducing the detection signal. This method had the characteristics of strong specificity and high sensitivity compared with the existing method due to the application of ligation-dependent probe signal recognition and amplification procedures. Under the optimized conditions, this method proved to be highly sensitive. The signal decreased as the concentration of copper ions increased, exhibiting a linear calibration from 0.03125 μM to 0.5 μM and a limit of detection of 18.25 nM. Subsequently, the selectivity of this biosensor was verified to be excellent by testing different relevant metal ions. Furthermore, this detection system of copper (II) ions was successfully applied to monitor Cu2+ contained in actual water samples, which demonstrated the feasibility of the biosensor.

Activity of nsp14 Exonuclease from SARS-CoV-2 towards RNAs with Modified 3'-Termini.

The COVID-19 pandemic has shown the urgent need for new treatments for coronavirus infections. Nucleoside analogs were successfully used to inhibit replication of some viruses through the incorporation into the growing DNA or RNA chain. However, the replicative machinery of coronaviruses contains nsp14, a non-structural protein with a 3'→5'-exonuclease activity that removes misincorporated and modified nucleotides from the 3' end of the growing RNA chain. Here, we studied the efficiency of hydrolysis of RNA containing various modifications in the 3'-terminal region by SARS-CoV-2 nsp14 exonuclease and its complex with the auxiliary protein nsp10. Single-stranded RNA was a preferable substrate compared to double-stranded RNA, which is consistent with the model of transfer of the substrate strand to the exonuclease active site, which was proposed on the basis of structural analysis. Modifications of the phosphodiester bond between the penultimate and last nucleotides had the greatest effect on nsp14 activity.

ACTIVITY OF NSP14 EXONUCLEASE FROM SARS-COV-2 TOWARDS RNAS WITH MODIFIED 3'-TERMINI

The COVID-19 pandemic has shown the urgent need for new treatments for coronavirus infections. Nucleoside analogs have been successfully used to inhibit replication of some viruses, through the incorporation into the growing DNA or RNA chain. However, the replicative machinery of coronaviruses contains nsp14, a a non-structural protein with a 3'→5'-exonuclease activity that removes misincorporated and modified nucleotides from the 3' end of the growing RNA chain. Here we studied the efficiency of hydrolysis of RNA containing various modifications in the 3'-terminal region by SARS-CoV-2 nsp14 exonuclease and its complex with the auxiliary protein nsp10. Single-stranded RNA was a preferable substrate compared to double-stranded, consistent with the model of transfer of the substrate strand to the exonuclease active site proposed on the basis of structural analysis. Modifications of the phosphodiester bond between the penultimate and last nucleotides had the greatest effect on nsp14 activity.

Self-powered DNA nanomachines for fluorescence detection of lead.

A versatile DNA nanomachine detection system has been developedvia the combination ofDNAzyme with catalytic hairpin assembly (CHA) technology for achieving accurate and sensitive detection of lead ions (Pb2+). In the presence of target Pb2+, capture DNA nanomachine formed by AuNP and DNAzyme recognized and reacted with Pb2+, which yielded an "active" DNAzyme, that induced the cleavage of substrate strand, and then released the initiator DNA (TT) for CHA. With the help of the initiator DNA TT, self-powered CHA was activated to achieve the signal amplification reaction in the detection of DNA nanomachine. Meanwhile, the initiator DNA TT was released and hybridized with the other H1 strand to initiate another CHA, replacement, and turnovers, producing enhanced fluorescence signal of fluorophore FAM (excitation 490nm/emission 520nm) for sensitive determination of Pb2+. Under the optimized conditions, the DNA nanomachine detection system revealed high selectivity toward Pb2+ in the concentration range 50-600pM, with the limit of detection (LOD) of 31pM. Recovery tests demonstrated that the DNA nanomachine detection system has excellent detection capability in real samples. Therefore, the proposed strategy can be extended and act as a basic platform for highly accurate and sensitive detection of various heavy metal ions.

Electrochemical determination of Pb2+ based on DNAzyme-triggered rolling circle amplification and DNA-templated silver nanoclusters amplification strategy

Lead ion (Pb2+) is one of the most widespread heavy metal contaminants that pose detrimental impact on environment and human health. An electrochemical biosensor was developed for detection of Pb2+ based on rolling circle amplification (RCA) of Pb2+-dependent DNAzyme and DNA-templated silver nanoclusters (DNA-AgNCs) mediated amplification strategy. The Pb2+-dependent DNAzyme (catalytic strand) hybridized with substrate strand to form a duplex structure. In the presence of Pb2+, DNAzyme was activated and cleaved substrate strand into two fragments at the “rA” site, releasing the enzyme strand as a padlock probe for subsequent RCA. The padlock probe hybridized with primer strand to form a circular template, which triggered RCA reaction in the presence of phi29 DNA polymerase and dNTPs. The resulted RCA product hybridized with DNA-AgNCs, catalyzing H2O2 to generate enhanced electrochemical signal. The electrochemical signal was proportional to the concentration of Pb2+ in the range of 1 pM−10 μM with a detection limit of 0.3 pM (S/N = 3). The proposed strategy exhibits highly sensitive and selective for Pb2+ analysis, providing potential application in real water samples and environment analysis.

The development of a paper-based distance sensor for the detection of Pb2+ assisted with the target-responsive DNA hydrogel

Due to the serious risks of lead pollution to human health, it plays a great role in constructing a simple, inexpensive, portable, and user-friendly strategy for Pb2+ detection in environmental samples. Herein, a paper-based distance sensor is developed to detect Pb2+ assisted with the target-responsive DNA hydrogel. Pb2+ can activate DNAzyme to cleave its substrate strand, which results in the hydrolysis of the DNA hydrogel. The released water molecules trapped in the hydrogel can flow along the patterned pH paper due to the capillary force. The water flow distance (WFD) is significantly influenced by the amount of water released from the collapsed DNA hydrogel triggered by the addition of various Pb2+ concentrations. In this way, Pb2+ can be quantitatively detected without using specialized instruments and labeled molecules, and the limit of detection (LOD) of Pb2+ is 3.0 nM. Additionally, the Pb2+ sensor works well in lake water and tap water. Overall, this simple, inexpensive, portable, and user-friendly method is very promising for quantitative and in-field detection of Pb2+ with excellent sensitivity and selectivity.

Carbon quantum dots@metal–organic framework based catalytic nucleic acid fluorescent system for highly sensitive and selective detection of Pb2+ in aqueous solutions

Accumulation of heavy metal ions in the ecosystem has become a major concern due to their adverse impacts on animals, humans, and aquatic habitats. The development of a rapid and portable sensing system is important for the prompt detection and proper management of such pollutants. Herein, we have designed a fluorescence-based biosensor for the selective and sensitive detection of Pb2+ ions using a DNAzyme system that is active only in the presence of lead (II) ions (Pb2+). The developed nanoprobe relies on two major components: (i) boron and nitrogen carbon dots (BNCDs)-doped carboxyl functionalized-terbium metal-organic framework (COOH-Tb MOF) as a fluorescent tag and (ii) quencher-modified catalytic NH3-GR5 DNAzyme as a bioreceptor molecule. The fluorescent tag (BNCDs/MOF) exhibits dual fluorescence with emission peaks located in the regions of blue and green. In the absence of Pb2+ ions, this hybridization undergoes reduced emission intensity because of the overlap in fluorescence emission between BNCDs@Tb-MOF and quenchers. However, as the catalytic core of the DNAzyme strand is activated in the presence of Pb2+ ions, the fluorescent emission of BNCDs/Tb-MOF resumes with the cleavage of quencher-tagged substrate strand. Here, the release of a shorter oligo sequence effectively increases the intensity of the proposed novel biosensor to realize low level detection of Pb2+ (e.g., to 0.96 ppb in a wide detection range from 2 to 1000 nM). The establishment of this novel biosensing approach is expected to offer new insights into rapid, selective, and sensitive detection of major pollutants for food and environmental safety.

A cell-surface-anchored DNA probe coupled with hybridization chain reaction enzyme-free dual signal amplification for sensitive electrochemical detection of the cellular microenvironment.

The cellular microenvironment plays key roles in regulating physiological processes. However, it is still a challenge to detect it with quantification. Here, a simple, biocompatible, and universal strategy based on cell surface-anchored specific DNAzymes and hybridization chain reaction enzyme-free signal amplification for cellular microenvironment electrochemical detection is presented. In this strategy, the cell could be captured on the surface of the electrode via aptamer-target recognition. On the other hand, the DNAzyme hybridized with the substrate strand as a metal ion probe was anchored on the surface of the cell. In the presence of metal ions, the substrate strand could be cleaved into two fragments by the DNAzyme and released from the cell surface. Then, the DNA modified gold nanoparticles (AuNPs) could be captured on the electrode. Subsequently, an alternative hybridization reaction of two hairpin probes was triggered by the carried initiators forming nicked double helices. For signal readout, hemin could be inserted into the double-helix DNA long chain via electrostatic interaction, which could electro-reduce hydrogen peroxide to generate an electrochemical signal. Based on the intrinsic advantages of DNAzymes, including rapid kinetics, high sensitivity, and high selectivity, and the signal amplification strategy, this method should be able to monitor and semi-quantify target metal ions in the cellular microenvironment. Furthermore, this method shows potential for various targets by employing different DNA probes in the cellular microenvironment, providing a platform for bioanalysis.

A fluorescent biosensor for Cd2+ detection in water samples based on Cd2+-fueled wheel DNAzyme walker and its logic gate applications

A fluorescent biosensor was developed for Cd2+ detection based on a Cd2+-fueled wheel DNAzyme walker. Cd2+ can activate the wheel to roll along the DNA walking tracks through DNAzyme cleavage and toehold-mediated strand displacement. The substrate strand was modified with BHQ and Cy5. Through continuous cleavage reactions toward the substrate strands, a high fluorescence signal can be obtained. The biosensor is ultrasensitive, and the detection limit is 0.2 pM (S/N = 3). The fluorescent assay is robust and has been applied to the determination of Cd2+ in real water samples with good accuracy and reliability. Using Cd2+, Pb2+, and Hg2+ as the three inputs, we also construct a concatenated AND logic gate. The input combination of (111) can produce an output of 1. Other input combinations produce an output of 0. Our proposed detection platform and logic system hold great promise for the ultrasensitive and intelligent sensing of different heavy metal ions in water samples.