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A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Cancer detection is still a major challenge in public health. Identification of oncogene is the first step toward solving this problem. Studies have revealed that various cancers are associated with miRNA expression. Therefore, the sensitive detection of miRNA is substantially important to solve the cancer problem. In this study, let-7a, a representative substance of miRNA, was selected as the detection target. With the assistance of magnetic beads commonly used in biosensors and self-synthesized graphene oxide materials, specificity and sensitivity detection of the target gene let-7a were achieved via protease-free signal amplification. The limit of detection (LOD) was as low as 15.015pM. The fluorescence signal intensity showed a good linear relationship with the logarithm of let-7a concentration. The biosensor could also detect let-7a in complex human serum samples. Overall, this fluorescent biosensor is not only simple to operate, but also strongly specificity to detect let-7a. Therefore, it has substantial potential for application in the early diagnosis of clinical medicine and biological research., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Continuous glucose monitoring is very important to daily blood glucose control in diabetic patients, but its accuracy is limited by the narrow linear range of the response of biosensor to the glucose concentration because of the oxygen starvation in tissue and the limited maximum conversion rate of glucose oxidase. In this work, a biocompatible diffusion limiting membrane based on two medical-grade polyurethanes is developed via blending modification to restrict the diffusion flux of glucose to match the oxygen concentration and the maximum conversion rate. The expansiveness of the linear range for the nanomaterials-modified electrode in the glucose biosensor can be achieved through the regulation of two polyurethanes, the solvent, and the thickness of the membrane. In addition, the mass transport of hydrogen peroxide and interfering substances is also limited of the membrane. The in vitro experiments demonstrated that the membrane-modified microneedle biosensor exhibited a rapid response to the concentration variation of glucose, a wide linear range that is sufficient to cover the blood concentration of healthy and diabetic people, the ability to resist the oxygen concentration fluctuation and interfering substances, good reproducibility and long-term stability. The custom wearable electrochemical system, possessing these characteristics, has been proven to accurately monitor the blood concentration in a living rat in real time. This demonstrates a significant potential for application in both daily and clinical blood glucose monitoring., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A nucleic acid aptamer based thermally oxidized porous silicon/zinc oxide microarray chip was constructed for the detection of ochratoxin A. The hybrid chains formed by aptamer and complementary chains labeled with fluorescent groups and fluorescent burst groups were used as recognition molecules, and the detection of toxins was accomplished on the chip by the principle of fluorescence signal burst and recovery. The modified QuEChERS method was used for sample pretreatment and the performance of the method was evaluated. The results showed that the linear range was 0.02 ∼ 200 ng/kg with the detection limit of 0.0196 ng/kg under the optimal detection conditions. The method was applied to different cereals with the recoveries of 90.30 ∼ 111.69 %. The developed microarray chip has the advantages of being cost-effective, easy to prepare, sensitive and specific, and can provide a new method for the detection of other toxins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The detection of miRNA in cells is difficult owing to its substantially low cellular content. Therefore, developing a highly sensitive sensor to detect cellular miRNA remains a significant challenge. Herein, we report an enzyme-assisted biosensor with target cycle amplification that can trigger the unlocking of locked hairpin probes for sensitive and robust let-7a gene detection. In the research, three kinds of hairpin probes were skillfully designed. The hairpin probe comprises a complementary sequence of a target, primer, and recognition site of Nt. BbvCI restriction endonucleases. In addition, the alternating synergistic impact of polymerase and the nicking enzyme generates considerable triggers to unlock the locked hairpin probe LH1, consequently triggering a subsequent circulating strand displacement reaction to form a stable H1-H2 double strand to ensure sufficient distance between a fluorophore on H1 and a quenching group on bolt DNA (bDNA), and resulting in the recovery of fluorescence. Furthermore, this process does not require complicated operation procedures and instruments, and the target gene let-7a can be sensitively detected. Specifically, the detection limit of the biosensor is as low as 160 fM, and its linear range is 0.5 pM-250 nM. Moreover, this biosensor can be employed to detect let-7a in human serum with good selectivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Frequency-mixing technology has been widely used to precisely identify magnetic nanoparticles in applications of quantitative biomedical detection in recent years. Examples include immune adsorption, lateral flow assays (LFAs), and biomagnetic imaging. However, the signals of magnetic response generated by adjacent magnetic samples interfere with each other owing to the small spacing between them in applications involving multi-sample detection (such as the LFA and multiplexing detection). Such signal interference prevents the biosensor from obtaining characteristic peaks related to the concentration of adjacent biomarkers from the magnetic response signals. Mathematical and physical models of the structure of sensors based on frequency-mixing techniques were developed. The theoretical model was verified and its key parameters were optimized by using simulations. A new frequency-mixing magnetic sensor structure was then designed and developed based on the model, and the key technical problem of signal crosstalk between adjacent samples was structurally solved. Finally, standard cards with stable magnetic properties were used to evaluate the performance of the sensor, and strips of the gastrin-17 (G-17) LFA were used to evaluate its potential for use in clinical applications. The results show that the minimum spacing between samples required by the optimized sensor to accurately identify them was only about 4-5 mm, and the minimum detectable concentration of G-17 was 11 pg mL -1 . This is a significant reduction in the required spacing between samples for multiplexing detection. The optimized sensor also has the potential for use in multi-channel synchronous signal acquisition, and can be used to detect synchronous magnetic signals in vivo., (© 2023 Wiley-VCH GmbH.)

A simple, rapid, low-cost, sensitive, intuitive, visual, label-free, colorimetric smartphone-assisted assay was developed for the measurement of aflatoxin B1 in miscellaneous beans. Ten different kinds of miscellaneous beans were treated and measured by modified QuEChERS(Quick、Easy、Cheap、Effective、Rugged、Safe) method with aflatoxin B1 nucleic acid aptamer as a recognition element and gold nanoparticles as indicators. Several factors influencing its sensitivity were investigated, including consumes and NaCl concentrations, as well as incubation time and specificity. The results showed a good linear relationship between concentrations of 0.2-8.0 ng/g under optimal conditions. With a detection limit of 0.08 ng/g, the linear regression equation was Y = 0.024X + 0.4615 (R = 0.9989). Sensor specificity is good. The content of aflatoxin B1 in bean samples was determined successfully. The recovery of aflatoxin B1 ranged from 87.18% to 110.24%. The whole thing took 15 min. This smartphone-assisted colorimetric aptamer sensor can be used to detect aflatoxin B1 in beans., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Herein, we have introduced hybridization chain reaction (HCR) into the photonic crystals (PhCs) hydrogel, for the first time, realizing HCR for inducing the change of the optical signal of PhCs hydrogel and using this hydrogel as a sensor for determination of the aflatoxin B1 (AFB1). By using specific sequences as the cross-linker, the extension of the cross-linker by HCR drives the swelling of the hydrogel, and the optical property of 2D PhCs array converts this swelling into a change of the Debye diffraction ring. Moreover, by further selecting the aptamer to construct the cross-linker, the hydrogel is also endowed with a unique capability for AFB1, making the hydrogel a novel sensor based on the signal amplification strategy. The results show that the designed hairpin DNAs can effectively trigger the HCR and cause the swelling of hydrogel, and the hydrogel sensor has a good determination performance and high specific recognition for AFB1., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) research in cancer diagnosis is expanding, on account of miRNAs were demonstrated to be key indicator of gene expression and hopeful candidates for biomarkers. In this study, a stable miRNA-let-7a fluorescent biosensor was successfully designed based on an exonuclease Ⅲ-assisted two-stage strand displacement reaction (SDR). First, an entropy-driven SDR containing a three-chain structure of the substrate is used in our designed biosensor, leading to reduce the reversibility of the target recycling process in each step. The target acts on the first stage to start the entropy-driven SDR, which generates the trigger used to stimulate the exonuclease Ⅲ-assisted SDR in the second stage. At the same time, we design a SDR one-step amplification strategy as a comparison. Expectly, this developed two-stage strand displacement system has a low detection limit of 25.0 pM as well as a broad detection range of 4 orders of magnitude, making it more sensitive than the SDR one-step sensor, whose detection limit is 0.8 nM. In addition, this sensor has high specificity across members of the miRNA family. Therefore, we can take advantage of this biosensor to promote miRNA research in cancer diagnosis sensing systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Herein, a ternary PdPtRu nanodendrite as novel trimetallic nanozyme was reported, which possessed excellent peroxidase-like activity as well as electro-catalytic activity on account of the synergistic effect between the three metals. Based on the excellent electro-catalytic activity of trimetallic PdPtRu nanozyme toward the reduction of H 2 O 2 , the trimetallic nanozyme was applied to construct a brief electrochemical immunosensor for SARS-COV-2 antigen detection. Concretely, trimetallic PdPtRu nanodendrite was used to modify electrode surface, which not only generated high reduction current of H 2 O 2 for signal amplification, but also provided massive active sites for capture antibody (Ab 1 ) immobilization to construct immunosensor. In the presence of target SARS-COV-2 antigen, SiO 2 nanosphere labeled detection antibody (Ab 2 ) composites were introduced on the electrode surface according sandwich immuno-reaction. Due to the inhibitory effect of SiO 2 nanosphere on the current signal, the current signal was decreased with the increasing target SARS-COV-2 antigen concentration. As a result, the proposed electrochemical immunosensor presented sensitive detection of SARS-COV-2 antigen with linear range from 1.0 pg/mL to 1.0 μg/mL and limit of detection down to 51.74 fg/mL. The proposed immunosensor provide a brief but sensitive antigen detection tool for rapid diagnosis of COVID-19., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Clinical diagnosis urgently requires ultrasensitive, accurate and rapid monitoring of low-abundance biomarkers. A biosensing strategy capable of detecting target genes at the femtomolar scale was designed in this work. In the biosensing strategy, the target can induce the specially designed hairpin probe H1 to self-fold and form a 3' blunt-ended structure. When there are the hybrid double-stranded P1-T1, ligase, polymerase and nickase, the target gene was recycled, and at the same time the system produces a lot of T1 and T2. T1 and T2 can simultaneously trigger HCR, causing the modified fluorophore FAM on the DNA strand to move away from the quencher group BHQ. The amplified fluorescent signal can be captured by a fluorescence instrument. It is exciting for us that three signal amplifications are involved to achieve femtomolar detection of target genes, namely target recycling, dual-triggered HCR of T1 and T2, and HCR. In addition, it still has good detection ability in actual samples simulated by serum. We expect that the sensing strategy proposed in this paper offers great potential for biomarker detection of leukemia for early clinical diagnosis., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Rapid and sensitive Escherichia coli (E. coli) detection is important in determining environmental contamination, food contamination, as well as bacterial infection. Conventional methods based on bacterial culture suffer from long testing time (24 h), whereas novel nucleic acid-based and immunolabelling approaches are hindered by complicated operation, the need of complex and costly equipment, and the lack of differentiation of live and dead bacteria. Herein, we propose a chemiluminescence digital microwell array chip based on the hydrolysis of 6-Chloro-4-methylumbelliferyl-β-D-glucuronide by the β-D-glucuronidase in E. coli to achieve fast single bacterial fluorescence detection. Taking the advantage of the picoliter microwells, single bacteria are digitally encapsulated in these microwells, thus the accurate quantification of E. coli can be realized by counting the number of positive microwells. We also show that the chemiluminescence digital microwell array chip is not affected by the turbidity of the test samples as well as the temperature. Most importantly, our method can differentiate live and dead bacteria through bacterial proliferation and enzyme expression, which is confirmed by detecting E. coli after pH and chlorination treatment. By comparing with the standard method of plate counting, our method has comparable performance but significantly reduces the testing time from over 24 h-2 h and 4 h for qualitative and quantitative analysis, respectively. In addition, the microfluidic chip is portable and easy to operate without external pump, which is promising as a rapid and on-site platform for single E. coli analysis in water and food monitoring, as well as infection diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this work, a brief electrochemical aptasensor was developed for highly sensitive detection of SARS-CoV-2 antigen utilizing an aptamer-binding induced multiple hairpin assembly strategy for signal amplification. In the presence of SARS-CoV-2, a pair of aptamers was brought in a close proximity according to the aptamer-protein antigen binding, which initiated strand displacement reaction thereby triggering a multiple hairpin assembly to obtain long linear DNA concatemers on the electrode surface. As the fabricated hairpin probes were labeled with biotin, massive streptavidin-alkaline phosphatases (ST-ALP) could be further introduced on the electrode interface via biotin-streptavidin interaction thus generating strong electrochemical signal in electrolyte solution containing 1-naphthol phosphate. Benefiting from the non-enzymatic multiple hairpin assembly signal amplification strategy, the designed aptasensor for SARS-CoV-2 spike protein detection exhibited the wide linear range from 50 fg·mL -1 to 50 ng·mL -1 and low detection limit of 9.79 fg·mL -1 . Meaningfully, this proposed electrochemical assay provided a potential application for the point of care analysis of viral diseases under ambient temperature., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Owing to the rapid increase in acute leukemia patients, the detection of Pax-5a, which is a tumor marker, is very important for the early diagnosis of patients. Therefore, by combining the selective digestion function of lambda exonuclease and the hybridization chain reaction (HCR) enzyme-free amplification system, we design a biosensor to detect the Pax-5a gene with high sensitivity. Lambda exonuclease can cleave the blunt end formed by the hairpin probe and the Pax-5a gene, which exposes the nucleic acid sequence that can initiate the HCR. When the HCR is triggered, the fluorophore and quencher on H1 and H2 move away from each other, so that the fluorescence signal of the quenched fluorophore can be recovered. Under optimal experimental conditions, a good linear relationship was established between the fluorescence intensity and the logarithm of the target concentration, and the limit of detection (LOD) of Pax-5a was calculated to be 7.6 pM. In addition, the biosensor can not only discriminate the base mismatch sequences of the Pax-5a gene, but also be suitable for target detection in complex human serum samples. Therefore, this biosensor, with the advantages of simple operation, high sensitivity, and good selectivity, has a good application prospect and guiding role in the diagnosis of acute B lymphocytic leukemia and the design of biosensors.

Lead ions are one of many common environmental pollutants, that can cause posing a serious threat to people's health, thus, their efficient and sensitive detection is important. We propose a cascade signal amplification sensor using a DNAzyme-based strand displacement amplification (SDA) and hybridization chain reaction (HCR) for the high-sensitivity detection of Pb 2+ . In the demonstrated sensor system, the target metal ion can activate DNAzyme to cause a strand displacement reaction. Under the synergistic action of polymerase and nickase, large numbers of DNA strands are generated that can initiate HCR. The subsequent HCR can restore the fluorescence intensity of the FAM quenched for the fluorescence resonance energy transfer effect, which exhibits a strong fluorescence signal. The DNAzyme-based sensor allowed the detection of Pb 2+ down to 16.8 pM and resulted in a good dynamic line relationship ranging from 50 pM to 500 nM, and the biosensor also showed good selectivity. Furthermore, we confirmed that the proposed sensor can still detect lead ions in complex environments such as lake water, milk, and serum. We believe these findings will provide new ideas for the detection of toxic elements ions in the environment and food., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Herein, a signal-on liquid crystal microarray (LCM) sensor is designed for the first time with a micro-spectral optical sensing signal. Depending on the change of the orientation of the LC molecules in the LCM films and the intensity of the spectral peaks of the PhCs, the signal-on LCM biosensor achieves the detection of AFB1 and the Partial Response Mechanism (PSM) of the LCM films is discovered.

One of the causes of B lymphocytic leukemia is abnormal expression of the Pax-5a gene. Detection of the Pax-5a gene can provide effective technical means for early screening of B lymphocytic leukemia. In this work, we designed a sensing scheme to detect the Pax-5a gene based on the signal amplification system, which is based on dual-enzyme assisted target gene circulation, and the disordered cleavage of CRISPR/Cas12a protease. The hairpin probe (HP) in this scheme not only contains the binding sites of the target gene and the primer, but also cleverly contains half of the Nt.BbvCI splicing sites. When the target gene is present, through the synergistic effect of KF and Nt.BbvCI, a large number of single strands of a specific sequence can be produced. At the same time, the target gene falls off from the first hairpin and opens the other hairpin to realize the cycle of the target gene. The resulting single-strand can bind to the Cas12a/crRNA binary complex and unlock the anti-cleavage activity of the CRISPR/Cas12a protease. The single strands labeled with the fluorescent group (FAM) and the quenching group (BHQ) around the solution are cleaved, the fluorescence signal of FAM is restored, and a detectable fluorescence signal is generated. The detection limit is as low as 6.77 fM, and the target gene and the mismatch sequence can also be distinguished well. Therefore, the sensing scheme provides a new detection direction for the early diagnosis and screening of B lymphocytic leukemia., (Copyright © 2022 Elsevier B.V. All rights reserved.)

It is of great significance to realize the detection of trace amounts of superoxide anions (O 2 • - ) in biological systems due to the connection between the concentration of O 2 • - and some diseases. Here, we present a novel method to fabricate a non-enzymatic electrochemical sensor for measuring O 2 • - from biological systems. The sensor was fabricated by nanocomposites (PAMAM-Au) synthesized by using (PAMAM-G4.0) dendrimers as template/stabilizers for gold nanoparticles (AuNPs) growth. Transmission electron microscopy (TEM) investigations indicate that AuNPs were encapsulated in PAMAM with average diameter about 8 nm. The electrochemical technique confirmed that PAMAM-Au possessed extraordinary electrochemical response to superoxide anions. As an analytical and sensing platform, apart from excellent sensitivity, wide liner range (from 3.69 × 10 -5 - 37.2 μM), ultra-low detection limit (0.0123 nM), our sensor also presented satisfying performance for monitoring changes of O 2 • - levels upon different stimuli. Furthermore, we investigated the effect of temperature on superoxide anions released from living cells and enzyme activity through an electrochemical method for the first time. Most importantly, the simulated electrochemical study in vitro further confirmed that temperature has a crucial influence on both the ability of living cells to resist external stimulation and the antioxidant capacity of superoxide dismutase. As expected, the study may provide researchers with potential value for understanding the temperature effects on organisms and the treatment of diseases caused by oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

In this work, Ti 3 C 2 T x MXene was identified as efficient nanozyme with area-dependent electrocatalytic activity in oxidation of phenolic compounds, which originated from the strong adsorption effect between the phenolic hydroxyl group and the oxygen atom on the surface of Ti 3 C 2 T x MXene flake. On the basis of the novel electrocatalytic activity, Ti 3 C 2 T x MXene was combined with alkaline phosphatase to construct a novel cascading catalytic amplification strategy using 1-naphthyl phosphate (1-NPP) as substrate, thereby realizing efficient electrochemical signal amplification. Taking advantage of the novel cascading catalytic amplification strategy, an electrochemical biosensor was fabricated for BCR/ABL fusion gene detection, which achieved excellent sensitivity with linear range from 0.2 fM to 20 nM and limit of detection down to 0.05 fM. This biosensor provided a promising tool for ultrasensitive fusion gene detection in early diagnosis of chronic myelogenous leukemia and acute lymphocytic leukemia. Moreover, the manageable catalytic activity of MXene broke a path for developing nanozymes, which possessed enormous application potential in not only electrochemical analysis but also the extensive fields including organic synthesis, pollutant disposal and so on., (© 2022. The Author(s).)

MicroRNAs (miRNAs) play a significant role in diverse biological processes. The abnormal expression of miRNAs is related to the development of cancers and various diseases. It is of great importance to sensitively and accurately detect miRNAs for early disease diagnosis and treatment. Here, a new fluorescence strategy was initially proposed for the enzyme-free sensing of let-7a by combining the strand displacement reaction (SDR) with the hybridization chain reaction (HCR). The sensor was successfully applied to the detection of the let-7a gene with a wide linear range from 25 pM to 250 nM and a limit of detection (LOD) of 9.01 pM. The fluorescence intensity has a good linear relationship with the logarithm of the target concentration. In addition, the biosensor allowed for the highly sensitive detection of the target genes even in complex human serum samples. With simple operation yet improved detection capability for let-7a, the developed fluorescent biosensor thus shows great potential for early clinical diagnosis as well as biological research., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Polarization of photoactive materials in current photoelectric (PE) systems is difficult to be adjusted, and thus electron-transfer routes of these systems are unchangeable, which limits their performance in photoelectrochemical (PEC) analysis. Herein, we attempted to modulate the polarization of perovskite-based heterostructures by both in situ semiconductor generation and enzyme catalysis. Owing to their band alignments, Cs 3 Bi 2 Br 9 quantum dots (QDs) and BiOBr are confirmed to construct a Z-scheme structure, leading to a large anodic photocurrent. In the presence of ascorbic acid 2-phosphate (AAP), BiPO 4 is generated on the surface of the Cs 3 Bi 2 Br 9 QDs/BiOBr heterostructure, reassigning energy bands of BiOBr. Accordingly, polarization of the photoactive materials is converted, and a new Z-scheme structure with a reversed electron-transfer route is constructed, which leads to an evident cathodic photocurrent. Furthermore, abundant electron donors can be obtained by catalyzing AAP with alkaline phosphatase (ALP). In this case, photogenerated holes in BiOBr are preferentially annihilated by electron donors, thereby blocking transfer of photogenerated electrons in the Cs 3 Bi 2 Br 9 QDs/BiOBr/BiPO 4 heterostructure. Consequently, a second polarization conversion is triggered by enzyme catalysis, resulting in the recovery of an anodic photocurrent. Benefited from the polarization conversion, a PEC biosensor with a feature of two-wing signal switch is designed, which remarkably enlarges the range of the signal response and subsequently improves the analytical performance. As a result, ALP in small volume of human serum can be quantified with this method. In this work, polarization of perovskite-based photoactive materials is tuned, proposing an alternative perspective on the design of advanced PE systems.

A design criterion to construct a versatile dual-modal colorimetric and PEC biosensing platform for switching the corresponding mode freely is proposed via integration of a natural enzyme, light-activated nanozyme and light-controlled swayable signal-output transverter. A switchable dual-modal platform toward DNA analysis is developed as a proof of concept.

Enzyme cascade sensors usually could not discriminate between the target and intermediate product. Herein, based on "AND" logic-controlled activation of the glucose oxidase-copper peroxide sensing system, enzyme cascade detection for glucose with resistance to inherently existing intermediate product H 2 O 2 was reported for the first time, which may provide a novel way for facilitating enzyme cascade sensing.

Since the discovery of the enzyme-like activities of nanomaterials, the study of nanozymes has become one of the most popular research frontiers of diverse areas including biosensors. DNA also plays a very important role in the construction of biosensors. Thus, the idea of combined applications of nanozymes with DNA (DNA-nanozyme) is very attractive for the development of nanozyme-based biosensors, which has attracted considerable interest of researchers. To date, many sensors based on DNA-functionalized or templated nanozymes have been reported for the detection of various targets and highly accelerated the development of nanozyme-based sensors. In this review, we summarize the main applications and advances of DNA-nanozyme-based sensors. Additionally, perspectives and challenges are also discussed at the end of the review.

Ratiometric fluorescent sensors are powerful tools for quantitative analyses. However, gold nano-clusters (AuNCs) as typical fluorophores in ratiometric sensors have some disadvantages, such as low luminous efficiency. In this study, a highly sensitive ratiometric fluorescence sensor was fabricated by the combination of AuNCs and fluorescein (FL), and the photonic crystals (PhCs) were used to selectively enhance the fluorescence intensity of AuNCs. This fluorescence sensor was used for the sensitive detection of acetylcholinesterase (AChE) and its inhibitor paraoxon. AChE can catalyze the hydrolysis of acetylthiocholine (ATCh) to form thiocholine (TCh), which can induce the fluorescence quenching of AuNCs while having no obvious influence on the fluorescence intensity of FL. AChE can be determined in the range from 0.1 to 25 mU mL-1 with a limit of detection (LOD) of 0.027 mU mL-1, and paraoxon can be determined in the range of 0.06 to 60 ng mL-1 with a LOD 0.025 ng mL-1. This method, as a new way to selectively improve the fluorescence signal of one of the fluorophores in the ratiometric sensor, would be a promising strategy for the sensitive determination of AChE and its inhibitor.

Here, we report a novel fluorescence method for the highly selective and sensitive detection of RNase H by combining the use of a dual-pyrene-labeled DNA/RNA duplex with supramolecular inclusion-enhanced fluorescence. Initially, the probe is in the "off" state due to the rigidness of the double-stranded duplex, which separates the two pyrene units. In the presence of RNase H, the RNA strand of the DNA/RNA duplex will be hydrolyzed, and the DNA strand transforms into a hairpin structure, bringing close the two pyrene units which in turn enter the hydrophobic cavity of a γ-cyclodextrin. As a result, the pyrene excimer emission is greatly enhanced, thereby realizing the detection of RNase H activity. Under optimal conditions, RNase H detection can be achieved in the range from 0.08 to 4 U/mL, with a detection limit of 0.02 U/mL., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Accurate and sensitive detection of the Pax-5a gene is of great importance in the early diagnosis and prognosis of acute leukaemia. Herein, a label-free electrochemical sensing system was proposed for the detection of the acute leukaemia Pax-5a gene based on enzyme-assisted signal amplification to generate abundant G-quadruplex/hemin DNAzyme. The presence of Pax-5a can open the hairpin probe (HP), which acts as a template. Under the action of the restriction enzymes Nt.BbvCI and Klenow fragment polymerase, the target gene Pax-5a is cycled to open the HP; On the other hand, a large number of G-quadruplex sequences are produced. The resulting G-quadruplex sequence is capable of forming the G-quadruplex/hemin complex on the surface of the electrode in the presence of hemin. The ultrasensitive label-free electrochemical detection of Pax-5a can be realized via the G-quadruplex/hemin complex-catalysed reduction of H 2 O 2 , and the detection limit was estimated to be as low as 4.6 fM. In addition, the biosensor has good specificity and stability, and also has excellent detection capabilities in a complex substrate environment. Therefore, the sensor shows great potential in bioanalysis and clinical diagnosis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The sensitive detection of tumor proto-oncogenes is indispensable because the early diagnosis and accurate treatment of genetic diseases is the key guarantee of patients' health. In this study, we proposed a novel palindromic molecular beacon (PMB) that it bases on the signal amplification strategy for ultrasensitive detection of Kras gene codon 12. PMB is designed to have two palindromic fragments at its two ends, one of which is locked via folding into a hairpin structure and the other promotes the formation of PMB duplex via intermolecular self-hybridization. Target DNA can hybridize to the loop portion of PMB and release the palindromic fragment at the 3' end. Within the PMB duplex, the two palindromic fragments released hybridize with each other and serve as polymerization primer responsible for the strand-displacement amplification (SDA). Namely, hybridized target DNA can be displaced and initiates the next round of reactions, making the polymerization/displacement/hybridization process go forward circularly. As a result, a large number of polymerization products are produced, dramatically enhancing optical signal. Because primer hybridization and polymerization-based displacement occur within PMB duplex, the reaction process is called intramolecular strand-displacement amplification (ISDA). Via utilizing the newly-proposed PMB-based ISDA strategy, the target K-ras gene could be detected down to 10 pM with a wide response range of 1 × 10 -11 -1.5 × 10 -7  M, and point mutations are easily distinguished, realizing the ultrasensitive, highly selective detection of K-ras gene. This impressive sensing paradigm demonstrates a new concept of signal amplification for the detection of disease-related genes only via using a simple way to efficiently amplify optical signal., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Silicon nanowire (Si NW) sensors have attracted great attention due to their ability to provide fast, low-cost, label-free, real-time detection of chemical and biological species. Usually configured as field effect transistors (FETs), they have already demonstrated remarkable sensitivity with high selectivity (through appropriate functionalisation) towards a large number of analytes in both liquid and gas phases. Despite these excellent results, Si NW FET sensors have not yet been successfully employed to detect single molecules of either a chemical or biological target species. Here we show that sensors based on silicon junctionless nanowire transistors (JNTs), the simplest possible transistors, are capable of detecting the protein streptavidin at a concentration as low as 580 zM closely approaching the single molecule level. This ultrahigh detection sensitivity is due to the intrinsic advantages of junctionless devices over conventional FETs. Apart from their superior functionality, JNTs are much easier to fabricate by standard microelectronic processes than transistors containing p-n junctions. The ability of JNT sensors to detect ultra-low concentrations (in the zeptomolar range) of target species, and their potential for low-cost mass production, will permit their deployment in numerous environments, including life sciences, biotechnology, medicine, pharmacology, product safety, environmental monitoring and security.

The demand of precise assay of nucleic acids and other bioanalytes has been increasing enormously in various areas including point-of-care diagnostics, military, environmental monitoring and so on. Compared with other nucleic acid biosensors, the electrochemical nucleic acid biosensors possess a range of merits like amenable miniaturization, low costs and high sensitivity. Ratiometric electrochemical nucleic acid biosensors can overcome the inherent systematic errors of conventional electrochemical biosensors and enhance the reproducibility and credibility. This short review (with 81 refs.) summarizes the evolvements made in the area of nucleic acid-based biosensors based on ratiometric (electrochemiluminescent, electrochemical and photoelectrochemical) readout in the past few years. Many of the methods discussed here are based on the use of advanced nanomaterials such as quantum dots, graphitic carbon nitrides, graphene oxide, C-dots, gold nanoparticles, metal-organic frameworks, and respective nanohybrids. Three sections (on electrochemiluminescence, classical electrochemical and emerging photoelectrochemical systems) demonstrate the merits of ratiometric assays in various applications. The review ends with a section with conclusions and a discussion of future perspectives. Graphical abstract Ratiometric sensing strategies overcome the intrinsic systematic errors of conventional electrochemical sensors that suffer from environmental and personal factors, and thus leads to remarkably enhanced reproducibility and reliability.

It is important to precisely monitor mercury (II) ions (Hg 2+ ) for environment protection and human health monitoring. Although many strategies have been developed in the past decades, there still remains a challenge for developing an ultrasensitive, simple and reliable approach to detect Hg 2+ . Herein, we report a ratiometric surface-enhanced Raman scattering (SERS) aptasensor by employing aptamer-modified Au@Ag core-shell nanoparticles (Au@Ag NPs) as highly functional sensing probes, allowing for ultrasensitive detection of Hg 2+ . In principle, the thiolated 5'-Cy3 labeled aptamer probe (Cy3-aptamer) is firstly immobilized on the SERS substrate surface and then hybridizes with the 5'-Rox labeled complementary DNA (cDNA) to form a rigid double-stranded DNA (dsDNA), in which the Cy3 and Rox Raman labels are used to produce the ratiometric Raman signals. In the presence of Hg 2+ , the aptamer DNA turns into the thymine (T)-Hg 2+ -T mediated hairpin structure, leading to the dissociation of dsDNA. As a result, the Rox labels are away from the Au@Ag NP SERS substrate while Cy3 labels are close to it. Therefore, the intensity of SERS signal from Cy3 labels increases while that from Rox labels decreases. The ratio between the Raman intensities of Cy3 labels and Rox labels is linear with Hg 2+ concentrations in the range from 0.001 to 1.0nM, and the limit of detection is estimated to be 0.4pM. The proposed strategy provides a new rapid, simple and reliable approach for sensitive detection of Hg 2+ and may create a universal methodology for developing analogous aptasensors for a wide range of other analytes determination., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Nanopatterning as a surface area enhancement method has the potential to increase signal and sensitivity of biosensors. Platinum-based bulk metallic glass (Pt-BMG) is a biocompatible material with electrical properties conducive for biosensor electrode applications, which can be processed in air at comparably low temperatures to produce nonrandom topography at the nanoscale. Work presented here employs nanopatterned Pt-BMG electrodes functionalized with glucose oxidase enzyme to explore the impact of nonrandom and highly reproducible nanoscale surface area enhancement on glucose biosensor performance. Electrochemical measurements including cyclic voltammetry (CV) and amperometric voltammetry (AV) were completed to compare the performance of 200 nm Pt-BMG electrodes vs Flat Pt-BMG control electrodes. Glucose dosing response was studied in a range of 2 mM to 10 mM. Effective current density dynamic range for the 200 nm Pt-BMG was 10-12 times greater than that of the Flat BMG control. Nanopatterned electrode sensitivity was measured to be 3.28 μA/cm 2 /mM, which was also an order of magnitude greater than the flat electrode. These results suggest that nonrandom nanotopography is a scalable and customizable engineering tool which can be integrated with Pt-BMGs to produce biocompatible biosensors with enhanced signal and sensitivity.

A novel microfluidic paper-based analytic device (μPAD) biosensor is developed for sensitive and visualized detection of glucose. This biosensor is easily fabricated using the wax printing technique, with a hybrid nanocomplex composed of dual enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) and Cu 3 (PO 4 ) 2 inorganic nanocrystals incorporated in the detection zones. The hybrid nanocomplex is found to exhibit a flower-like structure, which allows co-immobilization of these two enzymes in a biocompatible environment. These nanoflowers not only preserve the activity and enhance the stability of the enzymes, but also facilitate the transport of the substrates between the two enzymes. The biosensor is demonstrated to enable rapid and sensitive quantification of glucose in the concentration range of 0.1-10 mM with a limit of detection (LOD) of 25 μM. It is also shown to be applicable to colorimetric quantitative detection of glucose in human serum and whole blood samples, implying its potential for clinical applications.

A novel biosensor for sensitively detecting potassium ion (K + ) has been developed based on fluorescent copper nanoparticles (Cu NPs). In our design, we employ a label-free single-strand DNA (ssDNA) that contains two parts. One is 3'-terminus structure-switching aptamers (SSAs) that can fold into G-quadruplex after binding with its target K + . The other is 5'-terminus poly thymine (polyT) which works as a template to construct fluorescent Cu NPs. After incubating with K + , the part SSAs go through target-induced conformational changes. Benifiting from the exceptional digestion ability of exonuclease I (Exo I), the G-quadruplexes display effective resistance to nuclease digestion, so that 5'-terminus polyT remains and the in situ formation of Cu NPs provides a turn-on fluorescent signal that is used to evaluate the concentration of K + . The recovery of the fluorescence intensity is linearly correlated with the K + concentration in the range of 0.05 to 1 mM with a detection limit of 0.05 mM. Compared with some methods, this assay is cost-effective and facile with high specificity. Meanwhile, this excellent strategy shows a great potentiality in other sensing approaches that can study the interaction between similar SSAs and different specific targets.

A simple, label-free, and visual photonic crystal-based β-lactamase biosensor was developed for β-lactam antibiotic and β-lactamase inhibitor in which the penicillinase (a β-lactamase) was immobilized on the pH-sensitive colloidal crystal hydrogel (CCH) film to form penicillinase colloidal crystal hydrogel (PCCH) biosensing film. The hydrolysis of penicillin G (a β-lactam antibiotic) can be catalyzed by penicillinase to produce penicilloic acid, leading to a pH decrease in the microenvironment of PCCH film, which causes the shrink of pH-sensitive CCH film and triggers a blue-shift of the diffraction wavelength. Upon the addition of β-lactamase inhibitor, the hydrolysis reaction is suppressed and no clear blue-shift is observed. The concentrations of β-lactam antibiotic and β-lactamase inhibitor can be sensitively evaluated by measuring the diffraction shifts. The minimum detectable concentrations for penicillin G and clavulanate potassium (a β-lactamase inhibitor) can reach 1 and 0.1 μM, respectively. Furthermore, the proposed method is highly reversible and selective, and it allows determination of penicillin G in fish pond water samples.

We have developed a novel DNAzyme-based liquid crystal (LC) biosensor with high sensitivity for L-histidine, which is based on L-histidine-mediated formation of DNA duplexes by cleaving DNAzyme using L-histidine, resulting in a remarkable optical signal. Firstly, an optimal amount of capture probe is bound to the glass slide, which changes the surface topology as little as possible and shows a zero-background for the sensing system. When the DNAzyme molecule is cleaved by the target, L-histidine, a partial substrate strand is produced, which in turn can hybridize with the capture probe, forming a DNA duplex. The DNA duplexes induce LC molecules to undergo a homeotropic-to-tiled transition, obtaining a remarkable optical signal. The results show that the DNAzyme-based LC biosensor is highly sensitive to L-histidine with a detection limit of 50 nM. Compared with previously reported multi-step amplified methods, this newly designed assay system for L-histidine has no amplified procedures with comparable sensitivity. This method is an unprecedented example of DNAzyme-based LC biosensor for small molecules, which has potential to offer a DNAzyme-based LC model used in various targets., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report here an efficient pyrene excimer signaling-based time-resolved fluorescent sensor for the measurement of biothiols (cysteine (Cys), homocysteine (Hcy), glutathione (GSH)) in human serum based on thymine-Hg(2+)-thymine (T-Hg(2+)-T) coordination chemistry and the inclusion interaction of cyclodextrin. The sensing mechanism of the approach is based on the competitive ligation of Hg(2+) ions by Hcy/Cys/GSH and T-T mismatches in a bis-pyrene-labeled DNA strand with the self-complementary 5' and 3' ends. The introduction of γ-cyclodextrin can provide cooperation for the molecular level space proximity of the two labeled pyrene molecules, moreover the hydrophobic cavity of γ-cyclodextrin can also offer protection for the pyrene dimer's emission from the quenching effect of environmental conditions and enhance the fluorescence intensity of the pyrene excimer. When the biothiols are not presented, the sensing ensemble is in the "off" state due to the long distance between the two labeled pyrene molecules resulted from the formation of a more stable T-Hg(2+)-T structure. While in the presence of biothiols, Hg(2+) interacts very strongly with thiol groups and the T-Hg(2+)-T structure is dehybridized, and then the pyrene excimer will be formed due to the self-complementary 5' and 3' ends of the DNA probe and the cooperation interaction of γ-cyclodextrin to pyrene dimer, thus resulting in switching the sensing ensemble to the "on" state. In the optimum conditions described, the linear concentration range of 1.0-100 μM with the limit of detection (LOD) of 0.36 μM for GSH was obtained. Moreover, due to the much longer lifetime of the pyrene excimer fluorescence than those of the ubiquitous endogenous fluorescent components, the time-resolved fluorescence technique has been successfully used for application in complicated biological samples., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Using platelet-derived growth factor B chain dimer (PDGF-BB) as the model target, a background current eliminated electrochemical aptameric sensing platform for highly sensitive and signal-on detection of protein is proposed in this paper. Successful fabrication of the biosensor depends on ingenious design of aptamer probe, which contains the aptamer sequence for PDGF-BB and the recognition sequence for EcoRI endonuclease. In the absence of PDGF-BB, the ferrocene labeled aptamer probe folds into a hairpin structure and forms a recognition site for EcoRI. By treatment with endonuclease, the specific and cleavable double-stranded region is cut off and redox-active ferrocene molecule is removed from the electrode surface, and almost no peak current is observed. When binding with target protein, the designed aptamer probe changes its conformation and dissociates the recognition double strand. The integrated aptamer probe is maintained when exposing to EcoRI endonuclease, resulting in obvious peak current. Therefore, a signal-on and sensitive sensing strategy for PDGF-BB detection is fabricated with eliminated background current. Under the optimized experimental conditions, a wide linear response range of 4 orders of magnitude from 20pgmL(-1) to 200ngmL(-1) is achieved with a detection limit of 10pgmL(-1). Moreover, the present aptameric platform is universal for the analysis of a broad range of target molecules of interest by changing and designing the sequence of aptamer probe., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Noble metal nanoparticles are currently of great interest because of their unique optical properties and potential applications in disease diagnostics and cancer treatment. In the present work, a discovery was reported that dsDNA with terminal thiols at its two ends could lie easily flat onto the gold nanoparticle (GNP) surface rather than cross linked different GNPs, indicating an unique self-assembly behavior of newly-designed molecules on GNPs. This could intensively stabilize gold nanoparticles against aggregation even at a high salt concentration. On the basis of this discovery, a novel light-up colorimetric sensing strategy was developed for the detection of p53 gene by combining with the cyclical nucleic acid strand-displacement polymerization (CNDP). For the described colorimetric system, GNPs require no any surface functionalization, and target recognition reaction and CNDP amplification could be conducted under the optimized conditions to achieve a high efficiency. The high detection sensitivity and desirable selectivity are achieved, and the potential practical application was demonstrated. Besides, this sensing system can function in a wide range of salts, making it a suitable platform to cooperate with many biological processes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A novel highly-sensitive liquid crystal (LC) biosensing approach based on target-triggering DNA dendrimers was developed for the detection of p53 mutation gene segment at the LC-aqueous interface. In this study, the mutant-type p53 gene segment was the target to trigger the formation of DNA dendrimers from hairpin DNA probes by hybridization chain reaction, and the latter as a 'signal enhancement element' further induced the LC reorientation from tilted to homeotropic alignment, resulting in a corresponding optical changes of LC biosensors from birefringent to honeycombed textures or dark framework. The distinct optical reorientational appearances can serve as a characteristic signal to distinguish target concentrations ranging from 0.08 nM to 8 nM. Moreover, these optical phenomena suggest that the LC reorientation is related to the electric-dipole coupling between the adsorbed DNA and LC molecules, the conformational constraints of DNA and the internal electric field induction upon hybridization. This label-free LC biosensing strategy can open up a new platform for the sensitive detection of specific DNA sequences and enrich the application scope of an LC biosensing technique., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cell membrane-anchored biochemical sensors that allow real-time monitoring of the interactions of cells with their microenvironment would be powerful tools for studying the mechanisms underlying various biological processes, such as cell metabolism and signaling. Despite the significance of these techniques, unfortunately, their development has lagged far behind due to the lack of a desirable membrane engineering method. Here, we propose a simple, efficient, biocompatible, and universal strategy for one-step self-construction of cell-surface sensors using diacyllipid-DNA conjugates as the building and sensing elements. The sensors exploit the high membrane-insertion capacity of a diacyllipid tail and good sensing performance of the DNA probes. Based on this strategy, we have engineered specific DNAzymes on the cell membrane for metal ion assay in the extracellular microspace. The immobilized DNAzyme showed excellent performance for reporting and semiquantifying both exogenous and cell-extruded target metal ions in real time. This membrane-anchored sensor could also be used for multiple target detection by having different DNA probes inserted, providing potentially useful tools for versatile applications in cell biology, biomedical research, drug discovery, and tissue engineering.

The screening of potential drugs specifically binding to polydeoxyadenosine (poly(dA)) has been of great interest in recent studies. We have developed a simple colorimetric strategy through the mechanism of target induced split G-quadruplex formation for detecting coralyne, a poly(dA)-binding drug with noticeable antitumor activity. Two DNA oligonucleotides containing a split G-quadruplex sequence and an adenine-rich sequence are used in our strategy. In the presence of coralyne, the adenine-rich sequences of two oligonucleotides are drawn into close proximity, resulting in the formation of a split G-quadruplex DNAzyme that catalyzes the generation of a colored product. The DNAzyme-based colorimetric assay for coralyne has a linear range of from 0.033 to 1.667 μM with a low detection limit of 16 nM. The developed method is simple, cost-effective and visible; it holds great potential for applications in drug screening.

A novel label-free DNAzyme molecular beacon (MBzyme) strategy was for the first time developed for colorimetric amplification detection of target nucleic acids. The MBzyme, which is designed to contain peroxidase-mimicking DNAzyme that is locked by a common hairpin, was engineered to form a catalytically active MBzyme through hybridizing with the target p53 DNA. The MBzyme is a multifunctional label-free probe that can act as the target recognition element, catalytic DNAzyme and the primer of polymerization. The target p53 DNA hybridization can induce the isothermal circular strand-displacement polymerization even without any chemical modification and other DNA sequences. This unique amplifying strategy leads to the generation of multiple numbers of active MBzyme molecules even if one hybridization event occurs, achieving a dynamic range of seven orders of magnitude and giving a detection limit down to 25 fM which is 3-5 orders of magnitude lower than those of related literature reports. These achievements might be helpful in the design of highly efficient enhancers for G-quadruplex-hemin DNAzymes to be applied on the fundamental research, biotechnology, and biomedical diagnosis., (Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.)

We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10fM to 1nM, and the detect limit was as low as 8fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A label-free fluorescent DNA biosensor has been presented based on isothermal circular strand-displacement polymerization reaction (ICSDPR) combined with graphene oxide (GO) binding. The proposed method is simple and cost-effective with a low detection limit of 4 pM, which compares favorably with other GO-based homogenous DNA detection methods.

The development of autonomous DNA machines and their use for specific sensing purpose have recently attracted considerable research attention. In existing autonomous machines, the target recognition process and signal transduction are separated from each other. This results in misunderstanding of the operation behavior, and the assay capability is compromised when serving as a sensing tool. In this communication, the integrated signal transduction-based autonomous aptameric machine, in which the recognition element and signal reporters are integrated into a DNA strand, is developed. This new machine can execute the in situ amplification of target binding-induced signal. The authentic operation behavior of autonomous DNA machine is discovered: the machine's products directly hybridize to the "track" rather than to the signaling probes. Along this line, the machine is employed to detect the cocaine in a more straightforward fashion, and improved assay characteristics (for example, the dynamic response range is widened by more than 500-fold) are achieved. Our efforts not only clarify the concept described in traditional autonomous DNA machines but also have made technological advancements that are expected to be especially valuable in designing nucleic acid-based machines employed in basic research and medical diagnosis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel fluorometric assay method based on target-induced signal on was developed for acetylcholinesterase (AChE) inhibitor with obviously improved detection sensitivity. In this method, the AChE molecules catalyzed the hydrolysis of acetylthiocholine (ATCl) to form thiocholine, which in turn can specifically react with fluorescent squaraine derivative, a specific chemodosimeter for thiol-containing compounds, resulting in fluorescence quenching and offering a low fluorometric background for the further detection of AChE inhibitor. In the presence of AChE inhibitor, the catalytic hydrolysis of ATCl is blocked, and then the squaraine derivative remains intact and shows signal-on fluorescence. The amount of the remaining fluorescent squaraine derivative is positively correlated with that of the AChE inhibitor in solution. This new designed sensing system shows an obviously improved sensitivity toward target with a detection limit of 5 pg mL(-1) (0.018 nM) for the AChE inhibitor, comparing favorably with previously reported fluorometric methods. To our best knowledge, this new method is the first example of fluorometric enzymatic assay for AChE inhibitors based on such a signal-on principle and using a specific reaction, which has potential to offer an effective strategy for the detection of AChE inhibitors.

DNAzymes have been widely applied as signal amplifiers for enzyme-free and highly sensitive detection of DNA. A few of them have also been employed for amplified detection of other biomolecules via a target-triggered assembly of split or mutated DNAzyme strategy. However, most of these designs adopt Mg(2+)-dependent DNAzyme as the catalytic unit, which suffered from low catalytic cleavage activity. Meanwhile, some DNAzymes with high catalytic activity are not suitable for these designs because the slight modification of the catalytic core might results in remarkably decreased or even no catalytic activity of these DNAzymes. On the basis of DNAzyme topological effect or the terminal protection of small-molecule-linked DNA, we developed two versatile sensing platforms for amplified detection of different biotargets. Since no modification is necessary for the catalytic core of the DNAzyme in these designs, they can employ any DNAzyme with high catalytic activity as amplified unit, which affords a high amplified efficiency for the sensing platform. A catalytic and molecular beacon design was further employed to realize the true enzymatic multiple turnover of DNAzyme. These designs together allow a high sensitivity for the biotargets, resulting in a detection limit of 20 pM, 0.2 U/mL, and 1 ng/mL for target DNA, DNA adenine methylation methyltransferase (Dam MTase), and streptavidin, respectively, much lower than previously reported biosensors. In addition, the proposed sensing strategy is versatile. By conjugating with various recognition units, it can be employed to detect a wide range of biotargets, varying from nucleic acids to proteins with high sensitivity.

A novel label-free fluorescent strategy for the detection of glutathione (GSH) and cysteine (Cys) is presented. The system consists of two single stranded DNA (ssDNA) with thymine-thymine (T-T) mismatches and used Hg(2+) as a mediator, and N-methyl mesoporphyrin IX (NMM) as the signal reporter. The assay is based on the competitive reaction of Hg(2+) with GSH/Cys and T-T mismatched double stranded DNA (dsDNA). In the absence of the target, two ssDNA containing T-T mismatches react with Hg(2+) to form a T-Hg(2+)-T dsDNA structure in the solution, which hampers the formation of a G-quadruplex structure. However, in the presence of the target, GSH/Cys reacts with Hg(2+) to keep DNA probes in a free single state, resulting in the effective formation of a G-quadruplex structure of the DNA probe (GP). Subsequently, due to the strong interaction between the G-quadruplex structure and NMM, fluorescence was greatly enhanced. This fluorescence strategy does not require any chemical modification, making the assay convenient and cost-effective. This method exhibited a linear relationship between peak fluorescence intensity and concentration of GSH in the range of 10-400 nM with a limit of detection (LOD) of 9.6 nM. A linear range for Cys detection was obtained in the concentration range of 10-500 nM with an LOD of 10 nM. Moreover, the proposed method worked well for the analysis of complex biological samples.

Engineered nucleic acid probes containing recognition and signaling functions find growing interest in biosensor design. In this paper, we developed a novel electrochemical biosensor for sensitive and selective detecting of Hg(2+) based on a bifunctional oligonucleotide signal probe combining a mercury-specific sequence and a G-quadruplex (G4) sequence. For constructing the electrochemical Hg(2+) biosensor, a thiolated, mercury-specific oligonucleotide capture probe was first immobilized on gold electrode surface. In the presence of Hg(2+), a bifunctional oligonucleotide signal probe was hybridized with the immobilized capture probe through thymine-mercury(II)-thymine interaction-mediated surface hybridization. The further interaction between G4 sequence of the signal probe and hemin generated a G4-hemin complex, which catalyzed the electrochemical reduction of hydrogen peroxide, producing amplified readout signals for Hg(2+) interaction events. This electrochemical Hg(2+) biosensor was highly sensitive and selective to Hg(2+) in the concentration of 1.0 nM to 1 μM with a detection limit of 0.5 nM. The new design of bifunctional oligonucleotide signal probes also provides a potential alternative for developing simple and effective electrochemical biosensors capable of detecting other metal ions specific to natural or artificial bases., (Copyright © 2012 Elsevier B.V. All rights reserved.)