As-proposed Strategy Research Articles

Silver vanadium oxide@water-pillared vanadium oxide coaxial nanocables for superior zinc ion storage properties

The Ag0.333V2O5@V2O5·nH2O coaxial nanocables with optimal ion/electron conductivity enable synergistic enhancement of zinc ion storage properties.

Paper-based fluorescent sensor via aggregation induced emission fluorogen for facile and sensitive visual detection of hydrogen peroxide and glucose

Hydrogen peroxide (H2O2), an important reactive oxygen species (ROS), is related to the oxidative stress in organisms, and plays important roles in a variety of cellular activities as well. So it is of crucial importance to develop sensitive and accurate sensing strategies to detect H2O2 in biological systems. Herein, by taking advantage of the unique emission characteristics of aggregation induced emission (AIE) fluorogens, we proposed a non-enzymatic fluorescence platform for facile and sensitive detection of H2O2, both in solution state using fluorescence spectrometer and on paper-based sensor via visual inspection. Through the reaction between L-cysteine and H2O2, the fluorescence of TPE-M-L, an AIE fluorogen formed between maleimide-functionalized tetraphenylethene (TPE-M) and L-cysteine, is quenched, and highly sensitive non-enzymatic H2O2 assay is readily carried out. The limit of detection (LOD) of 10nM in solution state and 2.5μM on paper-based sensor were obtained for H2O2 detection, which were superior or comparable to those previously reported in literature. Moreover, by integrating glucose oxidase with the AIE fluorogen of TPE-M-L, highly sensitive and selective glucose detection was also conveniently achieved both in solution state and on paper-based sensor by the as-proposed strategy, with the LODs of 50nM in solution state and 10μM via visual observation, much better than those obtained by other fluorescence methods. The as-proposed sensing strategy was also successfully applied to assay glucose in human serum samples. Therefore, the paper-based fluorescence sensor exhibits the advantages of simple fabrication, high sensitivity and portability, and has great potential to be applied in on-site assay of H2O2 and glucose in real samples.

Integrating DNA structure switch with branched hairpins for the detection of uracil-DNA glycosylase activity and inhibitor screening

The detection of uracil-DNA glycosylase (UDG) activity is pivotal for its biochemical studies and the development of drugs for UDG-related diseases. Here, we explored an integrated DNA structure switch for high sensitive detection of UDG activity. The DNA structure switch containing two branched hairpins was employed to recognize UDG enzyme and generate fluorescent signal. Under the action of UDG, one branched hairpin was impelled folding into a close conformation after the excision of the single uracil. This reconfigured hairpin could immediately initiate the polymerization/nicking amplification reaction of another branched hairpin accompanying with the release of numerous G-quadruplexes (G4s). In the absence of UDG, the DNA structure switch kept its original configuration, and thus the subsequent polymerization/nicking reaction was inhibited, resulting in the release of few G4 strands. In this work, Thioflavin T was used as signal reporter to target G4s. By integrating the DNA structure switch, the quick response and high sensitivity for UDG determination was achieved and a low detection limit of 0.0001U/mL was obtained, which was superior to the most fluorescent methods for UDG assay. The repeatability of the as-proposed strategy was demonstrated under the concentration of 0.02U/mL and 0.002U/mL, the relative standard deviation obtained from 5 successive samples were 1.7% and 2.8%, respectively. The integrated DNA structure switch strategy proposed here has the potential application for the study of mechanism and function of UDG enzyme and the screening the inhibitors as potential drugs and biochemical tools.

Amphiphile-Mediated Ultrasmall Aggregation Induced Emission Dots for Ultrasensitive Fluorescence Biosensing.

The development of ultrasensitive and highly selective fluorescence biosensors for diverse analytes is highly desirable but remains a challenge. It is attributable to the scarcity of fluorogens with promising brightness, stability, and nontoxicity, which primarily determine the performance of fluorescence biosensors. Herein, we report the design and preparation of aggregation induced emission (AIE) dots with high brightness, exceptional colloidal stability, ultrasmall size, and functional groups for developing ultrasensitive biosensor through the electrostatic conjugation to biological molecules, and use blemycin (BLM) as the proof-of-concept analyte. The recognition and the subsequent cleavage of the quencher-labeled DNA (Q-DNA) by BLM result in the formation of three-mer quencher-linked oligonucleotide fragments (Q-DNA-1), which significantly decreases the amount of quenchers anchored on AIE dot surfaces and subsequently reduces the fluorescence resonance energy transfer (FRET) effect. As compared to the case in which BLM is absent, remarkable fluorescence enhancement is observed, and is dependent on BLM concentration. Thus, ultrasensitive fluorescence detection of target BLM is realized, with a detection limit down to 3.4 fM, the lowest value reported so far. Moreover, the proposed fluorescence biosensor has also been successfully utilized for detection of BLM spiked in human serum samples. The as-proposed strategy not only significantly improves the selectivity and sensitivity of BLM assay, but also allows the ultrasensitive detection of a variety of bioactive molecules by simply changing the specific target recognition substances, thus providing a versatile fluorescence platform, and showing great potential to be applied in chemo-/bioanalysis and clinical biomedicine.

A general strategy to facilely design ratiometric electrochemical sensors in electrolyte solution by directly using a bare electrode for dual-signal sensing of analytes

In this paper, we have described a general strategy to facilely design ratiometric electrochemical sensors in electrolyte solutions, directly using a bare electrode for dual-signal sensing of analytes. Two types of substances (methylene blue/MB, doxorubicin/DOX) with different electrochemical signal peaks were added into electrolyte solutions (phosphate buffered saline, NaCl), where one was the analyte (DOX) and the other was used as a reference (MB). A linear plotting of DOX concentration [DOX] versus ratiometric electrochemical signal peak intensity (IDOX/IMB) was achieved, with a good linear coefficient and low detection limit of DOX (0.4nM). Experimental results implied that this ratiometric electrochemical sensor (ECS) of DOX enabled highly selective and sensitive detection of DOX in real samples, with high detection recoveries. In comparison with previous reports about ratiometric ECS, this as-proposed strategy can directly fabricate a ratiometric ECS in electrolyte solution (not on electrode), only using a bare electrode for dual- signal sensing of analytes. This strategy is not only novel and facile, but also flexible and general, as adequately confirmed in experiments, which would facilitate a further development in the facile fabrication and efficient applications of electrochemical sensors.

A facile, sensitive, and highly specific trinitrophenol assay based on target-induced synergetic effects of acid induction and electron transfer towards DNA-templated copper nanoclusters

Reliable, selective and sensitive approaches for trinitrophenol (TNP) detection are highly desirable with respect to national security and environmental protection. Herein, a simple and novel fluorescent strategy for highly sensitive and specific TNP assay has been successfully developed, which is based on the quenching of the fluorescent poly(thymine)-templated copper nanoclusters (DNA-CuNCs), through the synergetic effects of acid induction and electron transfer. Upon the addition of TNP, donor-acceptor complexes between the electron-deficient nitro-groups in TNP and the electron-donating DNA templates are formed, resulting in the close proximity between TNP and CuNCs. Moreover, the acidity of TNP contributes to the pH decrease of the system. These factors combine to dramatically quench the fluorescence of DNA-CuNCs, providing a “signal-off” strategy for TNP sensing. The as-proposed strategy demonstrates high sensitivity for TNP assay, and a detection limit of 0.03μM is obtained, which is lower than those reported by using organic fluorescent materials. More significantly, this approach shows outstanding selectivity over a number of TNP analogues, such as 2,4,6-trinitrotoluene (TNT), 2,4-dinitrotoluene (DNT), 2,4-dinitrophenol (DNP), 3-nitrophenol (NP), nitrobenzene (NB), phenol (BP), and toluene (BT). Compared with previous studies, this method does not need complex DNA sequence design, fluorescent dye labeling, or sophisticated organic reactions, rendering the strategy with additional advantages of simplicity and cost-effectiveness. In addition, the as-proposed strategy has been adopted for the detection of TNP in natural water samples, indicating its great potential to be applied in the fields of public safety and environmental monitoring.

Biphasic photoelectrochemical sensing strategy based on in situ formation of CdS quantum dots for highly sensitive detection of acetylcholinesterase activity and inhibition.

Herein, we reported a facile and highly sensitive biphasic photoelectrochemical (PEC) sensing strategy based on enzymatic product-mediated in situ formation of CdS quantum dots (QDs), and assayed the activity and inhibition of acetylcholinesterase (AChE) in its optimal state. Upon the hydrolysis of acetylthiocholine catalyzed by AChE, the product thiocholine stabilizes the in situ formation of CdS QDs in homogenous solution. Due to the electrostatic attraction, the resulting tertiary amino group-functionalized CdS QDs are attached to the surface of the negatively charged indium tin oxide (ITO) electrode, generating significant PEC response upon illumination in the presence of electron donors. By taking full advantage of the in situ formation of CdS QDs in homogenous solution, this strategy is capable of detecting AChE activity and inhibition in its optimal state. A directly measured detection limit of 0.01mU/mL for AChE activity is obtained, which is superior to those obtained by some fluorescence methods. The inhibition of AChE activity by aldicarb is successfully detected, and the corresponding IC50 is determined to be 13μg/L. In addition to high sensitivity and good selectivity, this strategy also exhibits additional advantages of simplicity, low cost and easy operation. To the best of our knowledge, the as-proposed strategy is the first example demonstrating the application of CdS QDs formed in situ for biphasic PEC detection of enzyme activity and inhibition. More significantly, it opens up a new horizon for the development of homogenous PEC sensing platforms, and has great potential in probing many other analytes.

A versatile label-free and signal-on electrochemical biosensing platform based on triplex-forming oligonucleotide probe

Nucleic acid and protein assays are very important in modern life sciences, and the recently developed triplex-forming oligonucleotide probes provide a unique means for biological analysis of different kinds of analytes. Herein, we report a label-free and signal-on electrochemical sensor for the detection of specific targets, which is based on the triple-helix structure formation between the hairpin molecular beacon and the capture probe through the intermolecular DNA hybridization induced by Watson-Crick and Hoogsteen base pairings. Upon the introduction of a specific target, the triple-helical stem region is dissembled to liberate the hemin aptamer, and a G-quadruplex− hemin complex can be formed in the presence of K+ and hemin on the electrode surface to give an electrochemical response, thus signaling the presence of the target. With the use of Human Immunodeficiency Virus type 1 (HIV-1) as a proof-of-principle analyte, we first demonstrated this approach by using a molecular beacon, which consists of a central section with the DNA sequence complementary to HIV-1, flanked by two arm segments. This newly designed protocol provides an ultrasensitive electrochemical detection of HIV-1 with a limit of detection down to 0.054 nM, and also exhibit good selectivity. Therefore, the as-proposed strategy holds a great potential for early diagnosis in gene-related diseases, and with further development, it could be used as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.

Ultrasensitive homogeneous electrochemical strategy for DNA methyltransferase activity assay based on autonomous exonuclease III-assisted isothermal cycling signal amplification.

DNA methylation catalyzed by methyltransferase (MTase) plays an important role in many biological processes, including gene transcription, genomic imprinting and cellular differentiation. Herein, a simple and novel homogeneous electrochemical strategy for ultrasensitive DNA MTase activity assay has been successfully developed, which is based on methylation-triggered exonuclease (Exo) III-assisted autonomous isothermal cycling signal amplification. A duplex DNA (P1-P2 hybrid) containing the methylation-responsive sequence is ingeniously designed. In the presence of DNA adenine methylation (Dam) methyltransferase (MTase), P1-P2 hybrid is methylated and subsequently recognized and cleaved by Dpn I endonuclease, which triggers the Exo III-catalyzed autonomous cycling cleavage processes. Therefore, a large amount of methylene blue-labeled mononucleotides are released, generating a significantly amplified electrochemical signal toward the Dam MTase activity assay. The directly measured detection limit down to 0.004 U/mL is obtained, which is one or two orders magnitude lower than that of the approaches reported in literature. Since this assay is carried out in homogeneous solution phase under isothermal condition and sophisticated probe immobilization processes are avoided, it is very simple and easy to implement. Due to its advantages of ultrahigh sensitivity, excellent selectivity and simple operation, the as-proposed strategy has great potential in the applications in DNA methylation related clinical practices and biochemical researches.

Enzyme-guided plasmonic biosensor based on dual-functional nanohybrid for sensitive detection of thrombin.

Rapid and sensitive methodologies for the detection of protein are in urgent requirement for clinic diagnostics. Localized surface plasmon resonance (LSPR) of metal nanostructures has the potential to circumvent this problem due to its sensitive optical properties and strong electromagnetic near-field enhancements. In this work, an enzyme mediated plasmonic biosensor on the basis of a dual-functional nanohybrid was developed for the detection of thrombin. By utilizing LSPR-responsive nanohybrid and anaptamer-enzyme conjugated reporting probe, the sensing platform brings enhanced signal, stability as well as simplicity. Enzymatic reaction catalyzed the reduction of Au(3+) to Au° in situ, further leading to the rapid crystal growth of gold nanoparticles (AuNPs). The LSPR absorbance band and color changed company with the nanoparticle generation, which can be real-time monitoring by UV-visible spectrophotometer and naked eye. Nanohybrid constructed by gold and magnetic nanoparticles acts as a dual functional plasmonic unit, which not only plays the role of signal production, but also endows the sensor with the function of magnetic separation. Simultaneously, the introduction of enzyme effectively regulates the programming crystal growth of AuNPs. In addition, enzyme also serves as signal amplifier owing to its high catalysis efficiency. The response of the plasmonic sensor varies linearly with the logarithmic thrombin concentration up to 10nM with a limit of detection of 200 pM. The as-proposed strategy shows good analytical performance for thrombin determination. This simple, disposable method is promising in developing universal platforms for protein monitoring, drug discovery and point-of-care diagnostics.

Highly sensitive homogeneous electrochemical assay for methyltransferase activity based on methylation-responsive exonuclease III-assisted signal amplification

DNA methylation catalyzed by methyltransferase (MTase) plays a critical role in many biological processes. In this paper, a novel and highly sensitive homogeneous electrochemical assay was developed for the detection of DNA MTase based on methylation-responsive exonuclease III-assisted signal amplification. Upon the action of MTase/endonuclease on hairpin probe 1 (HP 1) containing the methylation-responsive sequence, single-stranded DNA segments are generated to hybridize with methylene blue (MB)-labeled hairpin probe 2 (HP 2). Then the digestion of HP 2 from the blunt 3′ terminus by exonuclease III is activated, resulting in the release of MB-labeled mononucleotides and the complementary DNA segment which could hybridize with another HP 2 to initiate the signal amplification process. The MB-labeled mononucleotide, due to its less negative charge and smaller size, diffuses easily to the negatively charged indium tin oxide (ITO) electrode, generating an amplified electrochemical signal. The detection limit of the proposed assay was estimated to be 0.04U/mL, which was better than or comparable to that of the biosensors previously reported. To the best of our knowledge, it is the first time to adopt exonuclease III-assisted signal amplification for homogeneous electrochemical assay of MTase activity, and this strategy exhibits the advantages of high sensitivity as well as simplicity. Since this assay is carried out in a homogeneous solution phase instead of on an electrode/solution interface, sophisticated probe immobilization processes could be avoided. The as-proposed strategy exhibits promising potential for MTase functional studies and related researches.

A signal-on electrochemiluminescence aptasensor based on the quenching effect of manganese dioxide for sensitive detection of carcinoembryonic antigen

We developed a signal-on electrochemiluminescence (ECL) aptasensor by using SI-ATRP to facilitate high-density immobilization of luminophores and manganese dioxide–graphene (MnO2–GO) composite to indirect deactivate the excited state of Ru(dcbpy)32+ for ultrasensitive detection of carcinoembryonic antigen (CEA). In this approach, manganese dioxide–graphene (MnO2–GO) composite served as an efficient quencher for indirect deactivating the excited state of Ru(dcbpy)32+. Surface initiated atom transfer radical polymerization (SI-ATRP) was applied to functionalize multiwalled carbon nanotubes (MWNTs) with glycidyl methacrylate (GMA) as the functional monomer. A nanocomposite material of polyamidoamine (PAMAM) dendrimer encapsulated AuNPs was used as the carrier to combine Ru(dcbpy)32+ and poly-GMA together for the synthesis of the ECL matrices. The prepared matrices were applied to bind amino-modified auxiliary probe I (A1), which was partially complementary with the CEA aptamer. Meanwhile, the MnO2–GO composite was modified with another amino-modified CEA aptamer-partial-complementary auxiliary probe II (A2). Through the hybridization of CEA aptamer with A1 and A2, the quencher MnO2–GO composite was linked with the ECL matrices, by which a low ECL signal was detected (off-state). However, in the presence of CEA, the sandwich-like structure was destroyed because CEA would bind to its aptamer in lieu of the auxiliary probes, which resulted in a recovery of ECL signal (on-state). The proposed ECL aptasensor showed high sensitivity with a detection limit of 25.3 fg mL−1 and a wide linear range of 0.1 pg mL−1 to 20 ng mL−1. Consequently, with the excellent sensitivity, stability and satisfying precision, the as-proposed strategy constitutes a promising detection technique for clinical diagnosis.

Autonomous exonuclease III-assisted isothermal cycling signal amplification: a facile and highly sensitive fluorescence DNA glycosylase activity assay.

One common form of DNA damage is the oxidation of guanine to 8-oxo-7,8-dihydroguanine (8-oxoG), which can be carcinogenic. Human 8-oxoguanine DNA glycosylase (hOGG1) is a key base excision repair (BER) enzyme that repairs 8-oxoG, and the expression level of hOGG1 is closely related to many types of human cancers. Herein, a novel and highly sensitive fluorescence biosensing platform for hOGG1 activity detection has been constructed based on autonomous exonuclease III (Exo III)-assisted signal amplification. Two hairpin probes (HP1 and HP2) are ingeniously designed. In the presence of hOGG1, HP1 is cleaved at the 8-oxoG site, and the stem is subsequently digested by Exo III, releasing the trigger DNA fragment (tDNA1). Successively, tDNA1 partially hybridizes with HP2 to initiate the Exo III-assisted cycling cleavage to release another trigger DNA fragment (tDNA2), which in turn triggers the cycling cleavage of DNA fluorescence probe (FP). Therefore, large amount of fluorophore fragments are released, leading to a significantly amplified fluorescence signal toward hOGG1 activity detection. A directly measured detection limit down to 0.001 U/mL is obtained, which is much lower than that of the approaches reported in literature. In addition to high sensitivity and good selectivity, the as-proposed strategy also exhibits the advantages of isothermal experimental condition, simplicity, and convenience. Furthermore, the Exo III-assisted autonomous cycling cleavage approach we proposed here is a universal sensing strategy and has great potential in assays of many other biological analytes.

Sensitive Nanochannel Biosensor for T4 Polynucleotide Kinase Activity and Inhibition Detection

5'-Polynucleotide kinase is a crucial class of enzyme that catalyzes the phosphorylation of nucleic acids with 5'-hydroxyl termini. This process regulates many important cellular events, especially DNA repair during strand damage and interruption. The activity and inhibition of nucleotide kinase have proven to be an evident effect on cellular nucleic acid regulation and metabolism. Here, we describe a novel nanochannel biosensor for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK), a famous member of the 5'-kinase family playing a major role in the cellular responses to DNA damage. On the basis of the functionalized nanochannel system and coupled λ exonuclease cleavage reaction, the nanochannel-sensing platform exhibits high sensitivity and convenience toward kinase analysis. Biotin-labeled dsDNA effectively blocks the streptavidin-modified nanochannel through forming a closely packed arrangement of DNA structure inside the channel. When dsDNA is phosphorylated by PNK and then immediately cleaved by λ exonuclease, the pore-blocking effect almost disappears. This PNK-induced microstructural distinctness can be directly and accurately monitored by the nanochannel system, which benefits from its high sensitivity to the change of the effective pore size. Furthermore, modification convenience and mechanical robustness also ensure the stability of the test platform. This as-proposed strategy exhibits excellent analytical performance in both PNK activity analysis and inhibition evaluation. The simple and sensitive nanochannel biosensor shows great potential in developing on-chip, high-throughput assays for fundamental biochemical process research, molecular-target therapies, and clinic diagnostics.