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As a kind of mycotoxin, aflatoxin B1 (AFB1), which is often found in agricultural products, poses a threat to human health. Developing a simple sensitive method for AFB1 detection is in great demand. Here, we reported an aptamer-based fluorescence assay for AFB1 detection by using DNAzyme to generate and amplify a signal. We redesigned a pair of DNA sequences, which originated from the anti-AFB1 aptamer and RNA-cleaving DNAzyme 10-23. In the absence of AFB1, the aptamer hybridized with the region of the substrate-binding arm of the DNAzyme, inhibiting the activity of the DNAzyme. In the presence of AFB1, the binding of AFB1 to the aptamer led to the displacement of the DNAzyme from the aptamer. The substrate-binding arm was unblocked, and the activity of the DNAzyme was restored for the hydrolysis of the fluorophore and quencher-labeled substrate, causing a significant fluorescence increase. This assay could detect AFB1 in the dynamic range from 0.98 to 2000 nmol/L with high selectivity, and the detection limit was 0.98 nmol/L. Moreover, the assay was able to detect AFB1 in a complex sample matrix. This work provides a useful tool for the analysis of AFB1., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Alpha-foetoprotein (AFP) is taken as a diagnostic tumor marker for the screening and diagnosis of cancer. Nucleic acid-based isothermal amplification strategies are emerging as a potential technology in early screening and clinical diagnosis of AFP. The leakages between hairpins dramatically increase the background and reduce the sensitivity. Thus, it is necessary to develop some strategies to reduce the leakage for isothermal amplification strategies. A DNAzyme-locked leakless enzyme-free amplification system was developed for AFP detection in liver cancer and breast cancer. AFP could open the apt-hairpin and initiate the catalytic hairpin assembly (CHA) reaction to produce a Y-shaped duplex. Two tails of a Y-shaped duplex cleaved the two kinds of leakless hairpins. Then, the third tail of the Y-shaped duplex catalyzed the second CHA between the cleaved leakless hairpins to recover the fluorescent intensity. The limit of detection reached 5 fg/mL by the two levels of signal amplifications. Importantly, the leakless hairpin design effectively reduced leakage between hairpins and weakened the background. In addition, it also showed a great promising potential for AFP detection in early screening and clinical diagnosis., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Herein, we subtly engineered an amplified colorimetric biosensor for the cyclic detection of African swine fever virus DNA (ASFV-DNA), which associated the branched catalytic hairpin assembly (bCHA) amplification with G-quadruplex DNAzyme activity through triplex DNA formation. Firstly, a Y-shaped hairpin trimer was constructed for the dynamic self-assembly of DNA dendrimers. Then, in the presence of ASFV-DNA, the signal strand CP was opened, exposing the toehold regions, which would trigger the CHA cascade reaction between hairpin trimers. In the CHA cascade reaction, H1, H2, and H3 opened and bound in sequence, eventually forming the structure of DNA dendrimers. Subsequently, the obtained bCHA product was specifically recognized by the GGG repeat sequences of L1 and L2, then amplified by the synergistic effect of triplex DNA and the formation of asymmetric split G-quadruplex. Benefiting from the amplification properties of bCHA and the high peroxidase-like catalytic activity of asymmetrically split G-quadruplex DNAzymes, it could achieve effective colorimetric signal output in the presence of ASFV-DNA by means of triplex DNA formation. Under the optimal experimental conditions, this biosensor exhibited excellent sensitivity with a detection limit of 1.8 pM. Further, it was applied to the content detection of simulated samples of African swine fever, and the recoveries were 98.9 ~ 103.2%. This method has the advantages of simple operation, good selectivity, and high sensitivity, which is expected to be used for highly sensitive detection of actual samples of African swine fever virus., (© 2023. Springer-Verlag GmbH Germany, part of Springer Nature.)

Based on a Pb 2+ -specific 8-17 DNAzyme-induced catalytic hairpin assembly (CHA), a simple signal-on fluorescence strategy for lead ion detection was established. 8-17 DNAzyme was used as the recognition element of Pb 2+ , which catalyzed the cleavage of the RNA base embedded in the DNA substrate strand, while releasing part of the substrate strand (S') as CHA initiator. And two hairpin probes (H1 and H2-FQ) were designed according to the sequence of S' for CHA, in which H2-FQ was labeled with the fluorophore FAM and quencher BHQ-1 as fluorescent "molecular switch" based on fluorescence resonance energy transfer (FRET). In the presence of Pb 2+ , the CHA reaction was triggered to form a large number of H1-H2 complexes, enabling enzyme-free isothermal amplification and a signal-on fluorescence strategy. In the concentration range of 0.5-1000 nM, the fluorescence signal increases with the increase of Pb 2+ concentration. The quantitative detection limit of Pb 2+ by this method is 0.5 nM, which has better detection performance compared with the FQ-labeled 8-17 DNAzyme method. The established biosensor exhibits good specificity and can be effectively used for the detection of Pb 2+ in real samples of river water and grass carp. Through ingenious nucleic acid sequence design, DNAzyme and CHA reactions are integrated to realize the enzyme-free isothermal amplifications and sensitive detection of Pb 2+ , which holds potential versatility in food supervision and environmental monitoring., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

The accurate and sensitive detection of survivin mRNA is of great significance for cancer diagnosis and treatment. However, limited by the low-abundance mRNA in live cells, most strategies of survivin mRNA detection that were one-to-one signal-triggered model (one target triggered one signal) were inapplicable in practice. Here, we reported a binding-induced DNAzyme motor triggered by the survivin mRNA, which was a one-to-more signal-triggered model (one target triggered more signals), amplifying the detection signal and enhancing the sensitivity. The nanomotor is constructed by assembling several DNAzyme motor strands silenced by the blocker strands, and dozens of FAM-labeled substrate strands on a single gold nanoparticle (AuNP), forming three-dimensional DNA tracks. Through building the survivin mRNA bridge between the blocker and the DNAzyme motor strand, the binding-induced DNA nanomotor could be triggered by survivin mRNA. The operation of the DNAzyme motor was self-powered. And each walking step of the DNAzyme motor was fueled by DNAzyme-catalyzed substrate cleavage, along with the cleavage of the fluorescent molecule, resulting in autonomous and progressive walking along the AuNP-based tracks, and the fluorescence increase. The DNAzyme motor exhibited excellent sensitivity and remarkable specificity for survivin mRNA, providing the potential for cell image., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

By utilizing a target biorecognition reaction to induce the self-assembly of G-quadruplexes and the aggregation of gold nanoparticles (Au NPs), this work develops a novel colorimetric biosensing method for kanamycin (Kana) antibiotic detection. The compact G-quadruplex structure was assembled from its two half-split sequences which were designed in two hairpin substrates of the Mg 2+ -dependent DNAzyme (MNAzyme). Besides hybridizing with the aptamer strand, the MNAzyme sequence was also split into two half fragments to be designed in the two substrates. Upon the aptamer-recognition reaction toward Kana, the MNAzyme strand could be quantitatively released to cause the exposure of the split G-quadruplex-sequences on two hairpin substrate-modified Au NPs and simultaneous release of two half fragments of the MNAzyme-sequence. Thus, the K + -assisted self-folding of G-quadruplexes causes the cross-linking of the two Au NPs to realize the Au NP aggregation-based colorimetric signal output (measured at the largest absorption peak near 520 nm). Meanwhile, the self-assembled formation of the second MNAzyme drastically amplified the signal response. Under the optimal conditions, a wide linear range from 0.1 pg mL -1  to 10 ng mL -1 and an ultrahigh sensitivity with the detection limit of 76 fg mL -1 were obtained. The dose-recovery experiments in real samples showed satisfactory results with recoveries from 98.4 to 105.4% and relative errors compared with the ELISA method less than 4.1%. Due to the high selectivity, excellent repeatability and stability, and simple manipulation, this method indicates a promising potential for practical applications. A novel homogeneous biosensing method was developed for the convenient detection of the kanamycin antibiotic. The target biorecognition-induced and dual DNAzyme-catalytic assembly of G-quadruplexes enabled the amplified aggregation of gold nanoparticles for the simple, cheap, stable, and ultrasensitive colorimetric signal transduction of the method., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A universal enzyme strand (E-DNA) recyclable L-histidine (L-His), melamine (MA), and cisplatin (CP) biosensor was fabricated on the basis of a target-specific RNA-cleaving DNAzyme with specific auramine O (AuO) dye instead of thioflavin T. In this strategy, the substrate strand (S-DNA) of the RNA-cleaving site was constructed as an intramolecular stem-loop structure, and a GT-rich sequence was imprisoned in the double-stranded stem which inhibits the formation of stable G-quadruplex (G4cpx) with AuO. The presence of L-His initiates a catalytic reaction for cleaving the RNA site of the S-DNA hydrolytically releasing the GT-rich portion, which subsequently combines with AuO and forms a G4cpx for enhanced fluorescent signal. The subsequent addition of MA uncoils the G4cpx to form T-MA-T dsDNA, or addition of CP unwound the G4cpx to form CP-DNA leading to an intensive decrease of AuO emission. Remarkably, the liberated L-His can ultimately cause several rounds of cleavage, and the liberated E-DNA can catalyze the subsequent reaction with the other S-DNA. The use of L-His and E-DNA repeatedly induces S-DNA cleavage and intensifies the emission signal. The results show that the proposed biosensor is extremely sensitive to L-His, MA, and CP with a detection limit of 0.98, 10, and 3.4 nM respectively. To the best of our knowledge, the utilization of AuO as the G4cpx inducer and stabilizer for L-His, MA, and CP detection in real milk and urine samples has never been reported., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

DNA is recognized as a powerful biomarker for clinical diagnostics because its specific sequences are closely related to the cause and development of diseases. However, achieving rapid, low-cost, and sensitive detection of short-length target DNA still remains a considerable challenge. Herein, we successfully combine the catalytic hairpin assembly (CHA) technique with capillary action to develop a new and cost-effective method, a target DNA- and pH-responsive DNA hydrogel-based capillary assay, for the naked eye detection of 24 nt short single-stranded target DNA. Upon contact of target DNA, three individual hairpin DNAs hybridize with each other to sufficiently amplify Y-shaped DNA nanostructures (Y-DNA) until they are completely consumed via CHA cycling reactions. Each arm of the resultant Y-DNA contains sticky ends with i-motif DNA structure-forming sequences that can be self-assembled in an acidic environment (pH 5.0) to form target DNA- and pH-responsive DNA hydrogels by means of i-motif DNA-driven crosslinking. When inserting a capillary tube in the resultant solution, the liquid level inside clearly reduces due to the decrease in capillary force induced by the gels. In this way, the developed assay demonstrates sensitive and quantitative detection, with a detection limit of approximately 10 pM of 24 nt short complementary DNA (cDNA) targeting SARS-CoV-2 RNA genes at room temperature within 1 h. The assay is further shown to successfully detect target cDNA in serum, and it is also applied to detect several types of target sequences. Requiring no analytic equipment, precise temperature control, or enzymatic reactions, the developed DNA hydrogel-based capillary assay has potential as a promising naked eye detection platform for target DNA in resource-limited clinical settings., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

MicroRNAs (miRNAs), found in blood and body fluids, have emerged as potential non-invasive biomarkers for disease and injury. miRNAs are quantitatively evaluated using typical RNA analysis methods such as the quantitative reverse transcription polymerase chain reaction, microarrays, and Northern blot, all of which require complex procedures and expensive reagents. To utilize miRNAs as practical biomarkers, it will be helpful to develop simple and user-friendly sensors. In this study, a paper-based miRNA sensor was developed by combining two methods: (1) target-recycled DNAzyme (Dz) amplification and (2) graphene oxide-assisted Dz blotting on paper. The Dz spots on paper caused a miRNA-dependent color change in presence of colorimetric reagents and facilitated the quantification of absolute amount of the target miRNA, irrespective of the volume, with high reproducibility. This approach is technologically straightforward and enables quantification of as low as 7.75 fmol miRNA using a portable smartphone., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

In this report, portable, quantitative, and sequential monitoring of copper ions and pyrophosphate (PPi) with a single sensor based on a DNAzyme-Fe 3 O 4 system and glucometer readout was performed. Initially, streptavidin was functionalized on the surface of magnetic Fe 3 O 4 spheres through glutaraldehyde. Then, an invertase-modified DNA Cu substrate was connected to the magnetic Fe 3 O 4 spheres by a specific reaction between streptavidin and biotin. The sensing system was formed by a hybridization reaction between the Cu substrate and Cu enzyme. In the presence of Cu 2+ , Cu 2+ will recognize the Cu DNA substrate and form an "off-on" signal switch, thereby resulting in the separation of invertase from the Fe 3 O 4 nanospheres. PPi recognizes Cu 2+ to form a Cu 2+ -PPi complex, resulting in an "on-off" signal switch. Under optimized conditions, linear detection ranges for Cu 2+ and PPi of 0.01-5 and 0.5-10 μM, and detection limits for Cu 2+ and PPi of 10 nM and 500 nM, respectively, were obtained. Good selectivity was achieved for the analysis of Cu 2+ and PPi. Satisfactory results were achieved for this biosensor during the determination of Cu 2+ in real tap samples and PPi in human urine samples. This verified that the sensor is portable and low cost, and can be applied to the sequential monitoring of multiple analytes with a single point-of-care biosensor., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

A colorimetric biosensor assay has been developed for Cd 2+ and Hg 2+ detection based on Cd 2+ -dependent DNAzyme cleavage and Hg 2+ -binding-induced conformational switching of the G-quadruplex fragment. Two types of multifunctional magnetic beads (Cd-MBs and Hg-MBs) were synthesized by immobilizing two functionalized DNA sequences on magnetic beads via avidin-biotin chemistry. For Cd 2+ detection, Cd-MBs are used as recognition probes, which are modified with a single phosphorothioate ribonucleobase (rA) substrate (PS substrate) and a Cd 2+ -specific DNAzyme (Cdzyme). In the presence of Cd 2+ , the PS substrate is cleaved by Cdzyme, and single-stranded DNA is released as the signal transduction sequence. After molecular assembly with the other two oligonucleotides, duplex DNA is produced, and it can be recognized and cleaved by FokI endonuclease. Thus, a signal output component consisting of a G-quadruplex fragment is released, which catalyzes the oxidation of ABTS with the addition of hemin and H 2 O 2 , inducing a remarkably amplified colorimetric signal. To rule out false-positive results and reduce interference signals, Hg-MBs modified with poly-T fragments were used as Hg 2+ accumulation probes during the course of Cd 2+ detection. On the other hand, Hg-MBs can perform their second function in Hg 2+ detection by changing the catalytic activity of the G-quadruplex/hemin DNAzyme. In the presence of Hg 2+ , the G-quadruplex structure in Hg-MBs is disrupted upon Hg 2+ binding. In the absence of Hg 2+ , an intensified color change can be observed by the naked eye for the formation of intact G-quadruplex/hemin DNAzymes. The biosensor assay exhibits excellent selectivity and high sensitivity. The detection limits for Cd 2+ and Hg 2+ are 1.9 nM and 19.5 nM, respectively. Moreover, the constructed sensors were used to detect environmental water samples, and the results indicate that the detection system is reliable and could be further used in environmental monitoring. The design strategy reported in this study could broadly extend the application of metal ion-specific DNAzyme-based biosensors., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

Testing multiple biomarkers, as opposed to one, has become a preferred approach for diagnosing many heterogeneous diseases, such as cancer and infectious diseases. However, numerous technologies, including gold standard ELISA and PCR, can detect only one type of biomarker, either protein or nucleic acid (NA), respectively. In this work, we report for the first time simultaneous detection of proteins and NAs in the same solution, using solely functional NA (FNA) molecules. In particular, we combined the thrombin binding aptamer (TBA) and the 10-23 RNA-cleaving DNA enzyme (DNAzyme) in a single aptazyme molecule (Aptazyme 1.15-3' ), followed by extensive optimization of buffer composition, sequences and component ratios, to establish a competitive bioassay. Subsequently, to establish a multiplex bioassay, we designed a new aptazyme (Aptazyme 2.20-5' ) by replacing the target recognition and substrate sequences within Aptazyme 1.15-3' . This designing process included an in silico study, revealing the impact of the target recognition sequence on the aptazyme secondary structure and its catalytic activity. After proving the functionality of the new aptazyme in a singleplex bioassay, we demonstrated the capability of the two aptazymes to simultaneously detect thrombin and NA target, or two NA targets in a multiplex bioassay. High specificity in target detection was achieved with the limits of detection in the low nanomolar range, comparable to the singleplex bioassays. The presented results deepen the barely explored features of FNA for diagnosing multiple targets of different origins, adding an extra functionality to their catalogue., (© 2021. Springer-Verlag GmbH Germany, part of Springer Nature.)

As an extremely important post-transcriptional regulator, miRNAs are involved in a variety of crucial biological processes, and the abnormal expressions of miRNAs are closely related to a variety of diseases. In this work, for the first time, we designed a nucleic acid lock nanostructure for specific detection of miRNA-21, which changes the self-structure to "active conformation" by binding the target, in order to generate triggers to initiate the subsequent reaction. Emphatically, this flexible nucleic acid lock is capable of self-cleaving without the assistance of external component, overcoming the disadvantages of the complex design and requiring protease assistance in traditional nanostructure. Moreover, the combination of DNAzyme and RCA technology not only greatly improves the efficiency of signal amplification but also enables primer generation to simultaneous cascade RCA amplification. Additionally, the electrochemical detection technology based on silver nanoclusters overcomes the shortcomings of traditional detection methods such as low sensitivity and complex operation. The detection limit achieved was 9.3 aM with a wide dynamic response ranging from 10 aM to 100 pM (at the DPV peak of - 0.5 V), which is comparable to most of the reported studies. Therefore, our work provided an ultra-sensitive way for the detection of miRNAs using nanostructures and revealed an effective means for disease theranostics and cancer diagnosis. In this work, for the first time, we designed a nucleic acid lock nanostructure based on its self-structural transformation for the specific detection of miRNA. And the combination of DNAzyme and cascade RCA reaction greatly improved the signal amplification efficiency., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

A portable biosensor has been developed based on microfluidic particle accumulation for visual quantification of copper ions. A copper-dependent DNAzyme is used to connect magnetic microparticles (MMPs) and polystyrene microparticles (PMPs), forming "MMPs-DNAzyme-PMPs." When copper ions are present, the DNAzyme is cleaved, allowing free PMPs to be released from the MMPs-DNAzyme-PMP complex. Using a capillary-flow-based microfluidic device, the MMPs-DNAzyme-PMPs are first separated by a magnetic chamber, allowing the free PMPs to continue flowing until being trapped at a particle dam with a narrowing nozzle. Therefore, as a thermometer-like display, the copper level can be visually quantified by the accumulation length of the free PMPs in the trapping microchannel. The limit of detection (LOD) is 33 nM determined by the linear range of 25-100 nM, which is 900 times lower than the prevalent standard (~30 μM) in Hong Kong. The system shows excellent selectivity (> 1000-folds) against other heavy metal ions and abilities to adapt to multiple water environmental conditions. Tests on tap water samples and three local natural water sources in Hong Kong manifest that the device can effectively monitor the quality of freshwater with >70% recovery and 26.16% RSD.

A sensing platform is presented for the determination of alkaline phosphatase (ALP) activity based on the cooperation of DNAzyme-Au spherical nucleic acid nanoprobe with the graphene-oxide-loaded hybridization chain reaction (HCR/GO) system to achieve good detection sensitivity and specificity. This assay takes advantage of the strong affinity of pyrophosphate (PPi) to Cu 2+ ions and the fact that ALP can hydrolyze pyrophosphate (PPi) to release free Cu 2+ ions. In the presence of ALP, the released Cu 2+ can promote the Cu 2+ -dependent DNAzyme to cleave the substrate that generates a shorter DNA fragment, which is responsible for further triggering the HCR/GO system to form a long fluorescence dsDNA and thereby giving an amplified fluorescence signal. Linear calibration range was obtained from 0.2 to 10 U L -1 , and the limit of detection (LOD) is about 0.14 U L -1 . The feasibility of the proposed method was validated by spiking ALP standards in bovine serum. The recovery ranged from 97.2 to 104.6%, and a coefficient of variation (CV) of less than 8% (n = 3) was obtained. This assay strategy was also applied to evaluate the ALP inhibitor efficiency, which indicates that the assay has potential for drug screening.

A DNAzyme-powered DNA walking machine was constructed to develop the fluorescence aptasensing for sensitive detection of kanamycin. The aptamer for kanamycin is partially hybridized with complementary DNA (cDNA) modified on magnetic beads (MBs). The specific interaction of target and aptamer triggered the cDNA to be free tentatively, which captured walker DNA. Then the autonomous motion of DNA walker on MBs surface was propelled via DNAzyme digestion of recognition sites. The signal probe was separated, and the amplified fluorescence signal was achieved by the accumulation of the signal probe. Kanamycin was used as a model analyte, and the developed assay achieves a detection limit of 0.00039 ng·mL -1 (S/N = 3) within a linear detection range from 0.001 to 2000 ng·mL -1 . This aptasensing strategy can be extended for detection of other antibiotics by adapting corresponding target recognition aptamer sequence. Graphical abstract The fluorescence aptasensing for sensitive detection of kanamycin based on DNAzyme-powered DNA walking machine was constructed.

Detection of lead(II) (Pb 2+ ) ions in water is important for the protection of human health and environment. The growing demand for onsite detection still faces challenges for sensitive and easy-to-use methods. In this work, a novel surface plasmon resonance (SPR) biosensor based on GR-5 DNAzyme and gold nanoparticles (AuNPs) was developed. Thiolated DNAzyme was immobilized on the gold surface of the sensor chip followed by anchoring the substrate-functionalized AuNPs through the DNAzyme-substrate hybridization. The coupling between the localized surface plasmon (LSP) of AuNPs and the surface plasmon polaritons (SPP) on the gold sensor surface was used to improve the sensitivity. The substrate cleavage in the presence of Pb 2+ ions was catalyzed by DNAzyme, leading to the removal of AuNPs and the diminished LSP-SPP coupling. The optimal detection limit was 80 pM for the sensor fabricated with 1 μM DNAzyme, corresponding to two or three orders of magnitude lower than the toxicity levels of Pb 2+ in drinking water defined by WHO and USEPA. By tuning the surface coverage of DNAzyme, the sensitivity and dynamic range could be controlled. This sensor also featured high selectivity to Pb 2+ ions and simple detection procedure. Successful detection of Pb 2+ ions in groundwater indicates that this method has the prospect in the onsite detection of Pb 2+ ions in water. Given the variety of AuNPs and metal-specific DNAzymes, this detection strategy would lead to the development of more sensitive and versatile heavy metal sensors. Graphical abstract.

Enabled by the coffee-ring effect, a paper-based signal transduce method is employed for catalytic hairpin assembly (CHA) amplification and hybridization chain reaction (HCR) to achieve miRNA quantification. Once the target miRNAs appeared, it was circularly used by CHA to initiate HCR amplification to produce a large number of G-quadruplex, which is combined with hemin to form a hemin/G-quadruplex DNAzyme. The DNAzyme catalyzes a colorimetric reaction to produce colored nanoparticles, which were converted to the end edge of the paper by evaporation-driven flow, forming a visible colored band. Higher concentration of miRNA led to more colored nanoparticles and thus a longer colored band that can simply be measured by a ruler. The results of determination of miRNA in samples demonstrate that the relative standard deviation of the proposed approach is 5.2%, highly sensitive and repeatable, with a working range 1.0 to 1000 pM and a LOD of 0.2 pM. The paper-based analytical device as a novel platform offers a new signal transduce pathway toward the detection of low-abundance biomarkers for diagnosis.Graphical abstract Schematic representation of the principle for quantification of miRNA on paper based on the coffee-ring effect.

A fluorescent biosensor based on the cascaded cyclic amplification-lighted copper nanoparticles has been developed, optimized, and validated. In the double-modular cascaded cyclic amplification, a DNAzymatic cyclic amplification unit transforms metal ion signal to specific DNA sequences, and a linear/exponential integrated amplification unit converts as-prepared DNA codes to identical thymine (T)-rich DNA templates. T-rich scaffolds can induce the generation of red fluorescent copper nanoparticles, with fluorescence emission at 625 nm upon the excitation at 340 nm, as signal vehicles for quantitative detection of metal ions. Copper ions, selected as the model target, could be detected in a wide linear range from 10 to 10 4  nM depending on the increased fluorescent intensity, and the detection limit is 5.6 ± 0.52 nM (n = 3) within 40 min, which is 4 orders of magnitude lower than the limits set in drinking water. In the detection of Cu 2+ in real tap and lake water, the results between inductively coupled plasma mass spectrometry (ICP-MS) and our proposed biosensor were consistent, illustrating the practicability of the fabricated method. In summary, the established fluorescent biosensor compensates the deficiency of immunoassays failing to analyze metal ions, broadens ranges of biomarkers responding to cleaved DNAzymes, provides an open platform sensing different metal ions, and meets the increasing need for the ultrasensitive detection in the field of food safety, environmental monitoring, and medical diagnosis.

Lead ions (Pb 2+ ), one form of the toxic heavy metal, have drawn significant attention due to their harmful effects on human health and the environment. Although many analytical techniques have been developed over the past few decades, the development of a sensitive, selective, and rapid method to detect Pb 2+ remains a challenge. In this work, we developed a sensitive surface-enhanced Raman scattering (SERS) biosensor for highly sensitive detection of Pb 2+ by using DNAzyme-modified Fe 3 O 4 @Au@Ag nanoparticles (Fe 3 O 4 @Au@Ag NPs). Firstly, the thiolated 5'-Cy3 DNA probe was modified on the surface of Fe 3 O 4 @Au@Ag NPs, which hybridized with the Pb 2+ -specific DNAzyme to form a SERS biosensor, and the Cy3 labels were used to detect Pb 2+ . In the presence of Pb 2+ , the DNAzyme cleaves the Cy3-labeled DNA probe, leading to the release of Cy3-labeled DNA probe from the Fe 3 O 4 @Au@Ag NPs. Therefore, the Raman intensity of the Cy3 labels decreases. The proposed biosensor exhibited excellent linearity in the range from 0.01 to 1.0 nM, with a limit of detection for Pb 2+ of 5 pM. It features superior selectivity to Pb 2+ over other interfering metal ions and good application in the determination of Pb 2+ in tap water and human serum samples. The SERS biosensor provides a novel' simple and sensitive method for detection of Pb 2+ and sheds new light on the design and synthesis of analogous SERS biosensors for the detection of other heavy metal ions.

In this work, a cloth-based chemiluminescence (CL) biosensor has been firstly presented for highly sensitive determination of long PCR amplicons. Under the action of a hybridization chain reaction, a good deal of hemin/G-quadruplex DNAzyme molecules are produced, which can effectively enhance the CL signal. Moreover, effective cloth-based DNA biosensors can be fabricated by sequential wax screen-printing and surface-modification processes. Especially, the integration of a desirable hydrophobic barrier and gravity/capillary flow onto the flow channel of the cloth-based device makes the biosensor easy to be fabricated and to be associated with a flow CL. For the luminol/H 2 O 2 -based CL system, the signals are triggered by the hemin/G-quadruplex DNAzyme and are recorded by a low-cost CCD. Under optimized conditions, the determination range of target DNA is 0.002-20,000 pM and its limit of detection is calculated to be 1.1 fM. The results show that the proposed CL biosensor has a good analytical performance, such as high detectability and specificity, wide linear range, and receivable reproducibility and stability. Finally, the proposed biosensor is proven by the fact that this method can successfully detect the target DNA prepared from the Listeria monocytogenes-spiked milk samples. Therefore, it is believed to have the potential application prospects in food safety and environmental monitoring. Graphical abstract.

A superior electrochemical biosensor was designed for the determination of UO 2 2+ in aqueous solution by integration of DNAzyme and DNA-modified gold nanoparticle (DNA-AuNP) network structure. Key features of this method include UO 2 2+ inducing the cleavage of the DNAzyme and signal amplification of DNA-AuNP network structure. In this electrochemical method, the DNA-AuNP network structure can be effectively modified on the surface of gold electrode and then employed as an ideal signal amplification unit to generate amplified electrochemical response by inserting a large amount of electrochemically active indicator methylene blue (MB). In the presence of UO 2 2+ , the specific sites on DNA-AuNP network structure can be cleaved by UO 2 2+ , releasing the DNA-AuNP network structure with detectable reduction of electrochemical response intensity. The electrochemical response intensity is related to the concentration of UO 2 2+ . The logarithm of electrochemical response intensity and UO 2 2+ concentration showed a wide linear range of 10~100 pM, and the detection limit reached 8.1 pM (S/N = 3). This method is successfully used for determination of UO 2 2+ in water samples. Graphical abstract Fabricated DNAzyme network structure for enhanced electrical signal. Numerical experiments show that the current signal decreases as the concentration of UO 2 2+ increases. It can be seen that the biosensors could be used to detect UO 2 2+ in aqueous solution effectively.

Based on the inner filter effect mechanism of quantum dots, a ratiometric fluorescence nanoprobe was constructed for the determination of Pb(II) ion. Green emitting quantum dots conjugated with DNA substrate (DNA 2 ) acted as donors providing green fluorescence, while gold nanoparticles coupled with DNA enzyme (DNA 1 ) as acceptors quench the green fluorescence. Meanwhile, Fe 3 O 4 nanosphere served as magnetic substrates to facilitate separation process and red fluorescence as an "inner rule" to eliminate the background signal. In the presence of Pb(II) ion, the DNA 1 specifically recognize and capture Pb(II) ion with enhanced catalytic activity, which can cleave DNA 2 and "turn on" the green fluorescence (I 540 ), while the red fluorescence (I 630 ) remained unchanged. In this way, the ratio of I 540 /I 630 reflects the Pb(II) ion in the system, enabling the quantitative and selective determination of Pb(II) ion over nine different metal ions. Under optimal conditions, the ratiometric fluorescence assay showed good linearity (R 2  = 0.98) within the range 10 to 100 ng mL -1 . The limit of detection (LOD) was calculated to be 1.79 pg mL -1 (S/N = 3, n = 3, ±3.8%). The proposed fluorescence nanoprobe provides better sensitivity and accuracy than non-ratiometric signal evaluation for Pb(II) ion determination. Schematic representation of ratiometric fluorescence nanoprobe for Pb(II) ion detection using green fluorescence of I 540 as "signal switch" and red fluorescence of I 630 as "inner rule." Graphical abstract.

The present study shows that a dual-signal nanoprobe consisting of DNAzyme-functionalized porous carbon nanospheres (PCNs) responds to microRNA-21 and zinc ion (Zn 2+ ). The fluorescent probe undergoes an increase in the fluorescence intensity of fluorescein isothiocyanate (FITC) (with excitation/emission wavelengths at 488/517 nm) and the fluorescence intensity of cyanine-5 (Cy5) (with excitation/emission wavelengths at 633/670 nm) in the presence of microRNA-21 and Zn 2+ . The recognition between microRNA-21 and its complementary strand in the PCNs induces the separation of Zn 2+ -specific DNAzyme from PCNs, thus resulting in the increase of green fluorescence, and the exogenous Zn 2+ triggers the rupture of cleavage strand of DNAzyme and recovery of red fluorescence. This nanoprobe allows us to acquire in vitro the determination of microRNA-21 in the range of 2-300 nM with a detection limit of 0.57 nM and the determination of Zn 2+ in the range 2-100 nM with a detection limit of 0.43 nM, and in situ simultaneous imaging in MCF-7 breast cancer cells. Therefore, this strategy permits to obtain the expression levels of different biomarkers in living cells, providing a useful tool for diagnosis of cancers and understanding their biological process. Graphical abstract Schematic representation of the DNAzyme-functionalized porous carbon nanospheres for the imaging analysis of microRNA-21 and Zn2+ in living cells.

RNA-cleaving DNAzyme is a very useful biomaterial for metal ions determination. However, parts of DNAzymes can be cleaved by several metal ions, which makes it difficult to distinguish the concentrations of different metal ions. A method was applied to determine the Cu(II) concentration by using electrochemical biosensors combined with a mathematical model. An electrochemical biosensor was fabricated using single carbon nanotubes/field-effect transistor (SWNTs/FET) functionalized with a DNAzyme named PSCu10 and its complementary DNA embedded phosphorothioate RNA (CS-DNA). The CS-DNA with amino groups at the 5' end was immobilized on the SWNTs' surface via the peptide bond and then combined with PSCu10 by identifying bases complementary pairing (Cuzyme/SWNTs/FET). The CS-DNA can be cleaved when Cu(II) bonded with the PSCu10 so that the structural change of Cuzyme improves the electrical conductivity of Cuzyme/SWNTs/FET. But CS-DNA also can be cut-off by the Hg(II) directly, which might interfere with the detection of the Cu(II) concentration using Cuzyme/SWNTs/FET. To solve this problem, Hgzyme/SWNTs/FET was employed to monitor the Hg(II) concentration at the same time, thus serving to determine the Cu(II) content through the Gaussian process regression. The biosensor array can determine the Cu(II) concentration varying from 0.01 to 10,000 nM when the Hg(II) concentration was ranging from 5 to 10,000 nM, and the limits of detection for Cu(II) and Hg(II) were 6.7 pM and 3.43 nM, respectively. Graphical abstract A biosensor array (Cuzyme/SWNTs/FET and Hgzyme/SWNTs/FET) was developed, and the detecting data were processed using Gaussian process regression. It allows determination of Cu(II) and Hg(II) concentrations with high accuracy.

A method is described for sensitive colorimetric determination of zinc(II) ions that uses (a) a Zn(II)-responsive hairpin DNAzyme that assists target recycling, (b) hybridization chain reaction, and (c) hemin/G-quadruplex DNA nanoladder. The Zn(II)-responsive split of the hairpin DNAzyme (HD) acts as the recognition and transformation probe. Upon addition of Zn (II) and enzyme strand, a duplex is formed in the loop region of hairpin. The caged initiator sequence is subsequently liberated from the HD by the Zn(II)-selective split of the substrate strand. This cleavage induces an enzyme strand recycling for the next round of cleavage. As a result, the initiator DNA is accumulated and cross-opened H1 and H2 to start a hybridization chain reaction (HCR). The caged G-quadruplex is released after the HCR to recruit hemin to form the hemin/G-quadruplex that is inserted into the DNA nanoladder. Once formed in the DNA nanoladder, these act as catalytic labels for the ABTS, resulting a green color change. This cascade amplification strategy allows 10 nM to 100 μM of Zn(II) to be linearly quantified by colorimetry at 415 nm with a detection limit of 3.5 nM. The recoveries ranged from 97.7 to 108.3% were obtained, confirming high reliability of the method for Zn 2+ analysis in lake water samples. Graphical abstractA colorimetric assay for Zn 2+ using hairpin DNAzyme (HD) assistant cycle and hemin/G-quadruplex lighted nanoladders is designed. A Zn 2+ -responsive split of HD is designed as the recognition and transformation probe. The hemin/G-Quadruplex inserted nanoladder act as reporter for signal readout.

A UV-vis, CD, and differential pulse voltammetric study was performed on the deactivation of the activity of parallel G-quadruplex/hemin DNAzymes (G4 DNAzymes) by Pb(II). The G4 DNAzyme carries a d[TC] sequence at its 3' end and is stabilized by potassium(I). On addition of Pb(II), the K(I) ions in the parallel G4 are replaced by Pb(II) to keep the parallel topology. Intruded Pb(II) decrease the affinity between the topology and hemin, this leads to a decrease of DNAzyme activity for catalyzing the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by hydrogen peroxide to form a green dye with an absorption maximum at 420 nm. The assay does not use any amplification, and has a linear response in the 0.01 to 10 μM Pb(II) concentration range and a 7.1 nM limit of detection. The method was successfully applied to the analysis of spiked water samples. Graphical abstractSchematic diagram of the colorimetric lead(II) assay based on the competition between K + and Pb 2+ stabilized G-quadruplex/hemin DNAzymes (G4 DNAzymes).

In this study, a sensitive amplification strategy for Pb 2+ detection using reduced graphene oxide (RGO) and gold nanoparticles (AuNPs) was proposed. Thiol-modified DNAzyme is specific for Pb 2+ self-assembly on RGO-AuNPs-modified electrode surface. Ferrocene labeled single-stranded DNAzyme (Fc-ssDNAzyme) self-hybridizes to form a DNA hairpin structure. The amount of Fc adsorbed on the electrode surface changes after the appearance of Pb 2+ , leading to a change of electrical signal. This change can be sensitively identified by differential pulse voltammetry (DPV) assisted by ferricyanide ([Fe(CN) 6 ] 3-/4- ) in the electrolyte. The high conductivity and specific surface area of RGO and the strong chemical bond adsorption effect between DNAzyme and AuNPs are responsible for the amplified detection of Pb 2+ , which realize a detection range of 0.05-400,000.0 nM and a minimum detection limit of 0.015 nM. Moreover, the selectivity test results indicated that the biosensor had specificity for Pb 2+ , even if there was interference from other high-concentration metal ions. This simple biosensor also exhibited good responsiveness in actual sample detection, which provides a good application prospect for field detection of Pb 2+ in water. Graphical abstract.

A voltammetric biosensor for lead(II) (Pb 2+ ) is described that is based on signal amplification by using an ion-dependent split DNAzyme and template-free DNA extension reaction. The Pb 2+ -dependent split DNAzyme was assembled on gold nanoparticles (Au@Fe 3 O 4 ), and this nanoprobe then was exposed to Pb 2+ which causes the split-off of DNAzymes to release primers containing 3'-OH groups (S 1 and S 2 ). The template-free DNA extension reaction triggers the generation of long ssDNA nanotails, which then can bind the free redox probe N,N'-bis(2-(trimethylammonium iodide)propylene)perylene-3,4,9,10-tetracarboxyldiimide (PDA + ) via electrostatic adsorption. Hence, the concentration of PDA + in solution is reduced. Therefore, less free PDA + can be immobilized on a glassy carbon electrode modified with electrodeposited gold nanoparticles (depAu) to produce an electrochemical signal, typically measured at ∼0.38 V (vs. SCE) for quantitation of Pb 2+ . The use of a Pb 2+ -dependent split DNAzyme avoids the usage of a proteinic enzyme. It also increases the sensitivity of the sensor which has a lower detection limit of 30 pM of Pb 2+ . Graphical abstract Novel electrochemical biosensor based on the amplification of ion-dependent split DNAzyme and template-free DNA extension reaction for trace detection of Pb 2+ .

A Pb(II)-DNAzyme is used in an amperometric method for the determination of Pb(II). The method is based on two feedback processes. In the first, the Pb(II)-DNAzyme initiates a reaction in presence of Pb(II) in a micro-tube to release a linear DNA (S 1 ). In the second, the S 1 triggers the recycling amplification between two types of hairpin-shaped DNA templates (H 1 and H 2 ) which consist of a primer sequence and a Pb(II)-DNAzyme substrate sequence. The Pb(II)-DNAzyme has excellent cleavage specificity toward the substrate sequence in S 1 that combined firstly with H 1 and then is linked to H 2 . This process will connect H 1 and H 2 . After hybridization with H 1 and H 2 to form two DNA complexes, S 1 is released and initiates the next recycling process. This results in efficient amplification. A glassy carbon electrode (GCE) was immersed into solution of HAuCl 4 to electrodeposit a layer of gold nanoparticles. This is followed by the assembly of the hairpin probe H 1 on the GCE. In addition, a nanohybrid consisting of 3, 4, 9, 10-Perylenetetracarboxylic acid (PTCA) and nitrogen-doped graphene (NG) was loaded with electroactive thionine (Thi) and gold to form nanoparticles of type NG-PTCA-Thi-Au. This is responsible for generating the amperometric signal (best measured at around -0.30 V vs. SCE) and also acts as the reducing agent for synthesizing the NG-PTCA-Thi-Au nanohybrid. H 2 is immobilized on NG-PTCA-Thi-Au to form a new tracer label. The concentration of Pb(II) in a solution can be quantified by determination of the amount of cleaved S 1 . The method has high sensitivity and selectivity for Pb(II). The detection limit is 0.42 pM (S/N = 3), and the detection range extends from 1 pM to 1000 nM. Graphical abstract Schematic representation of electrodes for the determination of the lead ions (Pb 2+ ). The sensor is using Pb 2+ -DNAzyme assisted recycling amplification based on hairpin assembly on a composite prepared from nitrogen doped graphene, perylenetetracarboxylic anhydride, thionine and gold nanoparticles (NG-PTCA-Thi-Au). This versatile platform expands studies on the detection of heavy metal ions.

A signal amplification method has been developed for determination of human papillomavirus-16 DNA (HPV-16 DNA). It consists of a combination of using (a) two-color emitting fluorescent DNA-templated silver nanoclusters (DNA-AgNCs); (b) hairpin-blocked DNAzyme probe (H-Dz), and (c) catalytic hairpin assembly. In the absence of the model target HPV-16 DNA, the H-Dz forms a hairpin structure through intramolecular hybridization. It inhibits the catalytic activity of the DNAzyme. However, in the presence of HPV-16 DNA, it will hybridize with the DNAzyme probe which leads to the opening of the hairpin and the formation of an active secondary structure in the catalytic core. This produces an "active" DNAzyme which can cut H-Dz under the catalytic action of Mg(II) ions. Then, the DNA fragment and HPV-16 DNA are separated due to the weak hybridization. The former triggers a downstream catalytic hairpin assembly (CHA). This changes the color of the fluorescence of DNA-AgNCs from red (630 nm) to yellow (570 nm). The HPV-16 DNA will hybridize with another H-Dz to trigger the next round of activation cycle to cut H-Dz. In this way, recycling amplification results in the generation of a strong signal that allows human HPV-16 DNA to be detected with a 5.7 pM detection limit. Graphical abstract A simple and effective cascade signal amplification method was developed for fluorometric determination of HPV-16 DNA. It is based on a combination of chameleon fluorescent DNA-AgNC, hairpin-blocked DNAzyme probe and catalytic hairpin assembly (CHA). Target recycling amplification results in significant signal changes, thus providing high detection sensitivity.

Rapid, portable, and efficient detection of lead cations (Pb 2+ ) is of great significance for monitoring and evaluating environmental toxicity and human healthcare. In this work, we developed a simple and low-cost homogenous fluorescence DNAzyme assay for Pb 2+ determination based on Pb 2+ -dependent cleaving and rolling circle amplification (RCA). DNAzyme and its substrate (5'-biotin) formed double-stranded hybrids in solution which could thoroughly react with Pb 2+ in aqueous phase. Then, the unreacted DNAzyme/substrate hybrids as well as cleaved parts with biotin labeling of substrate strand would be captured by magnetic beads through biotin-streptavidin interactions and removed from the reaction solution. Meanwhile, the other parts of the substrate strand remained in solution and subsequently acted as the primer and triggered RCA. The concentration of the cleaved substrate strand correlated to that of Pb 2+ and non-specific amplification was effectively minimized through biotin-streptavidin isolation. With a smartphone camera, the fluorescence intensity was recorded and quantified after 30-90 min amplification, making it a portable method with minimum instrumentation. A dynamic range of 1-100 nM of Pb 2+ was achieved under optimized conditions and it was successfully employed for Pb 2+ detection in spiked lake water. Graphical abstract.

A DNA dendrimer was constructed by combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR). A fluorescence resonance energy transfer (FRET) pair consisting of 6-carboxyfluorescein (FAM) and 5-carboxytetramethylrhodamine (TAMRA) was then created as a signaling probe for the ultrasensitive detection of Pb 2+ . Firstly, the DNAzyme released by Pb 2+ -induced recycling amplification as a primer induces RCA to open two different hairpins for generating repeated "Y" structures. Next, two other hairpins (labeled with FAM and TAMRA, respectively) are opened by the "Y" structures to trigger the HCR. As a result, a DNA dendrimer is generated. It is high loading with FRET pair and also promotes FRET pair mutually close to produce a remarkable FRET signal. Hence, ultrasensitive detection of Pb 2+ is accomplished by measurement of the ratio of the yellow fluorescence of TAMRA (peaking at 588 nm) and the green fluorescence of FAM (at 525 nm). The method works in the 0.001 to 10 nM Pb 2+ concentration range and has a 0.3 pM detection limit. It was applied to determination of intracellular Pb 2+ with convincing performance. Graphical abstractSchematic representation of novel DNA dendrimer produced by the combination of rolling circle amplification (RCA) and hybridization chain reaction (HCR) for immobilization of the fluorescence resonance energy transfer (FRET) pair as signal probe for sensitive determination of Pb 2+ .

An amplified electrochemical biosensing scheme is described for lead(II) ions. It is making use of DNAzyme-assisted target recycling and catalytic hairpin assembly (CHA). The hairpin strand (substrate probe for the Pb 2+ -based DNAzyme; referred to as SP) is composed of trigger probe (TP) and a capture probe 1 attached to gold nanoparticles (AuNP). In the presence of the enzyme probe that partially hybridizes with SP, the introduction of Pb 2+ triggers target recycling and drives the highly amplified translation of target Pb(II) to TP. The CHA reaction is further initiated by TP. The modified AuNP act as the connecting unit, and this leads to the formation of a 3D DNA-AuNP network on the electrode (which is the third amplification step). It can bind the positively charged redox mediator RuHex via electrostatic interaction for electrochemical detection. This biosensor has a low detection limit (95 pM) and any analytical range that covers the 100 pM to 5 μM Pb(II) concentration range. It is selective over other divalent metal ions. It was applied to the determination of Pb 2+ in spiked real-world samples. Graphical abstract Schematic presentation of the electrochemical biosensor. The triply amplified electrochemical assay is based on the use of DNAzyme-assisted target recycling with catalytic hairpin assembly (CHA) reaction for sensitive and selective determination of lead ion (Pb 2+ ). AuNP: gold nanoparticles; SP: substrate probe; EP: enzyme probe.

A sandwich-type electrochemical aptasensor has been constructed and applied for sensitive and selective detection of the carcinoembryonic antigen (CEA). The surface of a glassy carbon electrode (GCE) was first modified with nitrogen-doped graphene and then gold nanoparticles and graphene quantum dots electrodeposited on it to obtain an architecture of type GQD/AuNP/NG/GCE. In the next step, the CEA-binding aptamer was immobilized on the modified GCE. Hemin intercalates in the amino-modified hemin aptamer to form a hemin-G-quadruplex (hemin-G4) DNAzyme. The amino modified CEA aptamer II is connected to hemin-G4 by glutaraldehyde (GA) as a linker to produce CEAaptamerII/GA/hemin-G4 (=ApII/GA/DNAzyme). Through a sandwich mode, the ApII/GA/DNAzyme bioconjugates are captured on the modified GCE. Subsequently, the hemin-G4 acts as peroxidase-mimicking DNAzyme and rapidly catalyzes the electroreduction of hydrogen peroxide. The quantitative determination of CEA was achieved by differential pulse voltammetry, best at a working potential of around -0.27 V vs. Ag/AgCl. Under optimized conditions, the assay has a linear response in the 10.0 fg mL -1 to 200.0 ng mL -1 CEA concentration range and a lower detection limit of 3.2 fg mL -1 . Graphical abstract Schematic presentation of a sandwich-type electrochemical aptasensor based on nitrogen doped graphene (NG), gold nanoparticles (AuNPs) and graphene quantum dots (GQDs) modified glassy carbon electrode, and the hemin-G4 DNAzyme for femtomolar detection of the carcinoembryonic antigen.

A nonenzymatic fluorometric assay is described for highly sensitive and selective detection of silver ion. It is making use of a controlled DNA assembly and an AND logic operation of a multiple-component DNAzyme (MNAzyme). It corresponds to an Ag(I)-responsive three-way junction (3-WJ) assembly. The tailored probes of the 3-WJ architecture were designed with complementary domains for subsequent assembly. Cytosine (C)-cytosine mismatches at one-way junction were set as the sensing element for Ag(I). Upon exposure to Ag(I) as an input, C-Ag(I)-C pairs are being formed. This enhances the binding energy between these separate probes and thus promotes the formation of a nanostructure that represents an AND logic assembly of MNAzyme with an amplified output signal. This results in an Ag(I)-induced increase in fluorescence which is measured best at excitation/emission maxima of 645/670 nm. The method displays high selectivity and sensitivity, has a 5 pM detection limit at 3σ and a dynamic range that extends from 10 pM to 100 nM. Graphical abstract Schematic presentation of a new fluorescence system for determining silver ion by making use of cytosine-Ag(I)-cytosine pair formation, precisely-controlled DNA assembly and "AND" logic operation of multiple components DNAzyme (MNAzyme).

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. Graphical abstract Schematic presentation of target detection: the DNAzyme cannot cleave the substrate strand when target is absent. Once the target is added, the active DNAzyme can cleave the substrate strand in the presence of Mg 2+ , resulting in significant fluorescence enhancement when the release of the caged G-quadruplex sequences binding with 2-[4-(dimethylamino)phenyl]-3,6-dimethylbenzothiazolium chloride (ThT).

The authors describe a fluorometric and resonance Rayleigh scattering dual-mode scheme for detection of the activity and inhibition of alkaline phosphatase (ALP). The method utilizes (a) CoOOH nanoflakes, which have high resonance Rayleigh scattering activity and can strongly adsorb ssDNA, and (b) Co(II)-dependent DNAzyme assisted signal amplification. ALP specifically catalyzes the hydrolysis of ascorbic acid-2-phosphate to produce ascorbic acid which reduces CoOOH nanoflakes to Co(II) ion. The Co(II)-dependent DNAzyme is then activated by Co(II) ion, and this results in the cleavage of a substrate labeled with both a fluorophore and a quencher. Following hydrolysis, fluorophore and quencher become separated and the fluorescence measured at excitation/emission wavelengths of 490/518 nm recovers, while the RRS signal at 405 nm decreases. The method works on the 0.2 to 2000 U L -1 ALP activity range, and the detection limit is 0.05 U L -1 . The method was used to validate the mechanism of the action of two classical ALP inhibitors (EDTA and Na 3 VO 4 ). Conceivably, it can also be applied to screen for ALP inhibitors. Graphical abstract A bioprobe composed of cobalt oxyhydroxide (CoOOH) nanoflakes as the substrate and carrier, and Co 2+ -dependent DNAzyme containing guanine ribonucleotide (rG) and uracil ribonucleotide (rU) for circularly signal amplification is used for fluorometric determination of alkaline phosphatase (ALP) activity.

An ultrasensitive voltammetric aptasensor was constructed to analyze cardiac troponin I (cTnI). It is based on DNA nanotetrahedron (NTH) linked dual-aptamer (dAPT) and magnetic metal organic frameworks (mMOFs) of type Fe 3 O 4 @UiO-66. Firstly, the DNA NTH linked dAPT (Tro4 and Tro6) were immobilized on a gold electrode for improving the capture efficiency of cTnI. The novel mMOFs Fe 3 O 4 @UiO-66 was then decorated by Au@Pt nanoparticles (Au@PtNPs), horseradish peroxidase (HRP), G-quadruplex/hemin (GQH) DNAzyme, and two types of aptamers to form signaling nanoprobes. In the presence of cTnI, an aptamer-protein-nanoprobe sandwich-type structure is formed. Afterward, the nanoprobes including enzyme, GQH DNAzyme and Fe 3 O 4 @UiO-66/Au@PtNP were utilized to catalyze the oxidation of hydroquinone by hydrogen peroxide for the electrochemical signals amplification, typically at a working potential of -0.1 V (vs. Ag/AgCl). The voltammetric signal increases linearly in the 0.01 to 100 ng·mL -1 cTnI concentration range, and the detection limit is 5.7 pg·mL -1 . Graphical abstract An ultrasensitive voltammetric aptasensor was constructed to analyze cardiac troponin I (cTnI) based on DNA nanotetrahedron linked dual-aptamer and magnetic metal organic frameworks of type Fe 3 O 4 @UiO-66. The results indicated the aptasensor has a wide linear response range (0.01 to 100 ng/mL) and low detection limit (5.74 pg/mL) for cTnI. GE: gold electrode; MCH: 6-Mmercapto-1-hexanol; HRP: horseradish peroxidase; HQ: hydroquinone; BQ: benzoquinone.

A new fluorometric method is delineated for the detection of RNase H activity by combining DNAzyme with reduced graphene oxide (rGO). In the absence of RNase H, the fluorescence of FAM-labeled probe is quenched due to the strong adsorption on the rGO. The presence of RNase H can release the active DNAzyme from the DNA-RNA chimeric strand. This triggers the cleavage of the signal probe at the rA site with the help of the cofactor Mg 2+ . The recycle cleavage can directly result in the amplified signal emitted by the FAM-labeled short fragment. The method allows the activity of RNase H to be detected in a linear range of 0.01 to 5 U·mL -1 . The detection limit of 0.018 U·mL -1 is calculated by the principle of three-time standard deviation over the blank signal. Then, RNase H-targeting natural compounds were screened for their inhibitory action. Among the investigated compounds, five were screened as RNase H inhibitors in a concentration-dependent manner, and 4 compounds were identified as activators. Finally, the method was reliably used for discriminating the difference of RNase H activity in human serum. It is found that RNase H activity was upregulated in patients with hepatitis C virus infection. Graphical abstract The schematic presentation of rGO-DNAzyme-based RNase H detection. RNase H triggers the active DNAzyme releasing from the DNA-RNA chimeric strand, which can cleavage probes to FAM-labeled short fragments and make the fluorescence signal cycle amplified.

This work reports on a method for determination of lead(II) ion in environmental water samples. A Pb 2+ -specific DNAzyme immobilized on magnetic beads was coupled to rolling circle amplification (RCA) and pH meter-based readout. On addition of Pb 2+ ion, it induces partial cleavage of the DNAzyme on the magnetic beads. The single-stranded DNA remaining on the magnetic beads is used as the primer to trigger the RCA reaction with the assistance of a circular DNA template, polymerase and dNTPs. This results in the formation of numerous oligonucleotide repeats on the magnetic bead. Subsequently, these repeats hybridize with glucose oxidase-labeled single-stranded DNA (GOx-ssDNA) to form a long concatamer containing tens to hundreds of GOx-ssDNA tandem repeats. The concatenated GOx molecules oxidize glucose, and this is accompanied by a drop in the local pH value. The pH values (vs. background signal) drop linearly when Pb 2+ concentrations increase from 1.0-100 nM, and the detection limit is 0.91 nM. The method displays good reproducibility, high specificity and acceptable accuracy. It was applied to the analysis of spiked water samples, and results compared favorably with those obtained by ICP-MS. Graphical abstract Schematic presentation of metal-ion-induced DNAzyme on magnetic bead (MB) with rolling circle amplification (RCA) for pH meter-based detection of lead(II) ion and by using a glucose oxidase (GOx)-labeled probe.

An upgraded nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) is proposed on the basis of the exonuclease and polymerase activity of T4 DNA polymerase and Mg 2+ -dependent DNAzyme-assisted and hemin/G-quadruplex DNAzyme-assisted cascade amplification strategies. Briefly, the hybridized DNA of the target WMV sequence, HP1, and P1 was recognized and nicked by nicking endonuclease Nb.BbvCI, and two DNA segments (P1-25 and P1-6) were produced. P1-25 was digested in the 3'→5' direction, and digestion was halted at the 3'-terminal G locus with the exonuclease activity of T4 DNA polymerase. When dNTP solution mix was added to the mixture, an intact enzymatic sequence of Mg 2+ -dependent DNAzyme was synthesized by T4 DNA polymerase, which hybridized with its substrate sequence in the loop segment of HP2 immobilized on a gold electrode and initiated the cleavage round. The caged G-quadruplex sequence was released and formed hemin/G-quadruplex-based DNAzyme, resulting in sharply increased electrochemical signals. A correlation between the differential pulse voltammetry signal and the concentration of target WMV sequence was obtained in the range from 50 fM to 1 nM, with 50 fM detection limit. Because the nicking and polymerization reactions are irreversible and share the same buffer, the cascade amplification strategy is an ultrasensitive and high-efficiency strategy, indicating potential for viral detection. Graphical abstract An upgrade nicking/polymerization strategy for ultrasensitive electrochemical detection of Watermelon mosaic virus (WMV) was proposed based on DNAzyme-assistant cascade amplification strategies.

A colorimetric assay for ATP is described that uses a strategy that combines the concept of split Mg(II)-dependent DNAzyme, split aptamer, and hybridization-induced aggregation of gold nanoparticles (AuNPs). Both ATP aptamer and Mg(II)-dependent DNAzyme are split into two fragments which are allocated to two well-designed DNA probes. The probes also possess mutually complementary stem sequences and spacer sequences. In the presence of ATP, the separated DNAzyme sequences in the two probes assemble via the synchronous recognition of ATP with two fragments of the aptamer. Then, the activated DNAzyme catalyzes multiple cycles of the cleavage of its substrate DNA sequence. The latter acts as a linker and induces the aggregation of two types of ssDNA-modified AuNP through the hybridization between the complementary sequences. Thus, the color of the AuNP solution remains red. However, in the absence of ATP, the detached aptamer cannot induce the assembly of DNAzyme to cleave the linker DNA. This results in the aggregation of AuNP and a concomitant color transition from red to purple. This ATP assay, performed at a wavelength of 530 nm, has a linear detection range that extends from 10 pM to 100 nM, with a detection limit of 5.3 pM. It was applied to the detection of ATP in human serum. Conceivably, the strategy has a wide scope in that it may be applied to the colorimetric detection of various other analytes through the split aptamer configuration. Graphical abstract Schematic presentation of colorimetric assay for adenosine triphosphate (ATP) based on the use of a split Mg(II)-dependent DNAzyme, a split aptamer, and by exploiting the hybridization-induced aggregation of gold nanoparticles that leads to a color change from red to purple.

DNA- and MNAzymes are nucleic acid-based enzymes (NAzymes), which infiltrated the otherwise protein-rich field of enzymology three decades ago. The 10-23 core NAzymes are one of the most widely used and well-characterized NAzymes, but often require elevated working temperatures or additional complex modifications for implementation at standard room temperatures. Here, we present a generally applicable method, based on thermodynamic principles governing hybridization, to re-engineer the existing 10-23 core NAzymes for use at 23 °C. To establish this, we first assessed the activity of conventional NAzymes in the presence of cleavable and non-cleavable substrate at 23 °C as well as over a temperature gradient. These tests pointed towards a non-catalytic mechanism of signal generation at 23 °C, suggesting that conventional NAzymes are not suited for use at this temperature. Following this, several novel NAzyme-substrate complexes were re-engineered from the conventional ones and screened for their performance at 23 °C. The complex with substrate and substrate-binding arms of the NAzymes shortened by four nucleotides on each terminus demonstrated efficient catalytic activity at 23 °C. This has been further validated over a dilution of enzymes or enzyme components, revealing their superior performance at 23 °C compared to the conventional 10-23 core NAzymes at their standard operating temperature of 55 °C. Finally, the proposed approach was applied to successfully re-engineer three other new MNAzymes for activity at 23 °C. As such, these re-engineered NAzymes present a remarkable addition to the field by further widening the diverse repertoire of NAzyme applications.

A catalytic cleavage strategy was developed for the fluorometric determination of Hg(II). The method is based on the use of a Mg(II)-dependent split DNAzyme. Fluorophore labeled hairpins were conjugated to gold nanoparticles (AuNPs) upon which fluorescence is quenched. Thymine-Hg(II)-thymine (T-Hg(II)-T) interaction causes the two DNA sequences to form an entire enzyme-strand DNA (E-DNA). The E-DNA bind to the hairpins on the AuNPs to form a Mg(II)-dependent DNAzyme structure. The circular cleavage of hairpins results in a signal amplification and in the recovery of fluorescence. The assay has a limit of detection (LOD) as low as 80 pM of Hg(II). This LOD is comparable to those obtained with other amplification strategies. The method was successfully applied to the determination of Hg(II) in Chinese herbs (Atractylodes macrocephala Koidz). Graphical abstract Schematic of a catalytic cleavage strategy based on Mg(II)-dependent split DNAzyme for fluorometric determination of Hg(II).

A fluorometric assay for zinc ion is described that relies (a) on the use of an isothermal cycle to amplify the fluorescence signal, and (b) of magnetic beads (MBs) to completely remove unreacted DNA detection probes. Biotin and fluorophore-labeled substrate (Zn-Sub) strands acting as detection probes were first assembled on MBs. Next, Zn(II)-specific DNAzyme (Zn-Enz) strands were hybridized with the Zn-Sub strands. In the presence of Zn(II), the Zn-Sub strands are cleaved. This results in the release of the shorter DNA fragments (containing fluorescent label) and in the dissociation of Zn-Enz strands. The dissociated Zn-Enz strands then hybridize with the residual Zn-Sub strands and cleave them in a similar fashion. This leads to a target recycling amplification mechanism and in a cumulative signal amplification process. A strongly amplified signal is thus obtained in the presence of Zn(II). The use of MBs warrants that unreacted Zn-Sub strands can be magnetically separated from the solution. The method has a detection limit as low as 33 fM at a signal-to-noise ratio of 3 and a linear response in the 100 fM to 11 nM Zn(II) concentration range. It was applied to the determination of Zn(II) in spiked tap water and seawater samples, and the results compared well with data obtained by ICP-MS analysis. The method was also applied to the determination of Zn(II) in infant milk powder and breast milk. Graphical abstract Magnetic beads (MBs) carrying fluorescein-labeled substrate (Zn-Sub) strands were hybridized with Zn(II)-specific DNAzyme (Zn-Enz) and cleaved in the presence of Zn(II). After recycling, the unreacted Zn-Sub strands were removed with MBs and the released fluorescein tags are measured.

A DNAzyme-embedded hyperbranched DNA dendrimer is used as a colorimetric signal amplifier in an ultrasensitive detection scheme for nucleic acids. The hyperbranched DNA dendrimers were constructed by single-step autonomous self-assembly of three structure-free DNA monomers. A cascade of self-assembly reactions between the first and second strands leads to the formation of linear DNA concatemers containing overhang flank fragments. The third strand, which bears a peroxidase-mimicking DNAzyme domain, serves as a bridge to trigger self-assembly between the first and second strands across the side chain direction. This results in a chain branching growth of the DNAzyme-embedded DNA dendrimer. This signal amplifier was incorporated into the streptavidin-biotin detection system which comprises an adaptor oligonucleotide and a biotinylated capture probe. The resulting platform is capable of detecting a nucleic acid target with an LOD as low as 0.8 fM. Such sensitivity is comparable if not superior to most of the reported enzyme-free (and even enzyme-assisted) signal amplification strategies. The DNA dendrimer based method is expected to provide a universal platform for extraordinary signal enhancement in detecting other nucleic acid biomarkers by altering the respective sequences of adaptor and capture probe. Graphical abstract Schematic of an assembly of a DNAzyme-embedded hyperbranched DNA dendrimer which operates as a signal amplifier for nucleic acids detection. The nanostructure is constructed by autonomous self-assembly of three DNA monomers. Colored letters represent each domain, and complementary domains are marked by asterisk. Domain d represents the DNAzyme sequence.

A one-step, one-tube colorimetric assay is described for the detection of bacterial double-stranded DNA (dsDNA). It utilizes a G-quadruplex DNAzyme produced by an asymmetric polymerase chain reaction (As-PCR) that catalyzes the oxidation of cysteine to form cystine. This results in the formation of oligonucleotide-modified gold nanoparticles via triplex formation, and eventually in a color change from red to blue that occurs within about 10 mins. This can be measured by ratiometric colorimetric (at 525 and 600 nm). The limit of detection (LOD) for the model analyte (dsDNA of Staphylococcus aureus (S. aureus)) is as low as 0.28 pg per 0.05 mL with a good linear response ranging from 16.0 fg·μL -1 to 1.6 ng·μL -1 . This is much lower than previously reported LODs. The assay is highly selective for S. aureus dsDNA over a range of other bacterial DNAs. Conceivably, it provides an attractive alternative tool for rapid detection of bacterial dsDNA as required in pathogen screening in the food industry. Graphical abstract Schematic presentation of a colorimetric assay for bacterial DNA. It is based on the catalytic activity of a G-quadruplex DNAzyme that is formed by an asymmetric PCR involving triplex DNA formation and gold nanoparticle (AuNPs) aggregation.

Radioactive radon decays into a stable daughter product, 210 Pb, which was used as the detection target to determine the radon radiation dose in a new technique. Pb 2+ triggers DNAzyme to cleave a molecular beacon (MB), resulting in the stem-loop structure opening and forming two single DNA strands (ssDNA). The ssDNA binds to unmodified gold nanoparticles and effectively prevents their aggregation in a salt solution. The detached enzyme strands continue to complement the remaining MB to amplify the response signal. The method proposed in this study exhibited a good linear relationship for Pb 2+ and radon concentrations in the range of 6.22 × 10 2 -1.02 × 10 5  Bq h/m 3 with a detection limit of 186.48 Bq h/m 3 using an ultraviolet-visible spectrometer. In practical applications, this sensitive method can avoid radioactive damage in field testing, and the detection limit meets the national standard in China. Importantly, this simple, highly sensitive strategy uses simple equipment and has a strong anti-interference ability. Graphical abstract.

The authors describe a fluorometric strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. This strategy relies on the combination of target-modulated photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA (labeled with silver nanoclusters) along with hairpin probe-based circular exponential amplification. The reaction system involves three hairpin probes (H1, H2 and H3). Probe H1 contains an aptamer against S. Typhimurium and the recognition sequence for nicking endonuclease. It is used to recognize S. Typhimurium and participates in polymerase-catalyzed target recycle amplification and secondary-target recycle amplification. Probe H2 contains an aptamer against hemin and is used to form the G-quadruplex DNAzyme in the presence of hemin and potassium ion. It acts as the electron acceptor and quenches the fluorescence of the labeled DNA. Fluorescence is best measured at excitation/emission wavelengths of 567/650 nm. Probe H3 contains the template sequence for the synthesis of AgNCs and the H2-annealing sequence. Both H2 and H3 are utilized to perform a strand displacement reaction and to achieve PET between G-quadruplex DNAzyme and DNA/AgNCs. To the best of our knowledge, this is the first example of a PET between G-quadruplex DNAzyme and DNA/AgNCs coupled with circular exponential amplification. The assay has an ultra-low detection limit 8 cfu·mL -1 of S. Typhimurium. The assay is rapid, accurate, inexpensive and simple. Hence, the strategy may represent a useful platform for ultrasensitive and highly specific detection of pathogenic bacteria as encountered in food analysis and clinical diagnosis. Graphical abstract The reaction system includes three hairpin probes (H1, H2 and H3), primer probe (P), Phi 29 DNA ploymerase (Phi 29) and nicking endonuclease Nt.AlwI (Nt.AlwI). Phi 29 and Nt.AlwI -assisted signal amplification leads to the recycling of target and produces numerous single stranded-DNAs (S). Strand displacement amplification leads to photoinduced electron transfer (PET) between G-quadruplex DNAzyme and DNA/AgNCs. HAP-based circular exponential amplification of PET results in an ultrasensitive fluorometric assay.