Taiping Qing

Poly(Thymine)-Templated Fluorescent Copper Nanoparticles for Ultrasensitive Label-Free Nuclease Assay and Its Inhibitors Screening

Noble-metal fluorescent nanoparticles have attracted considerable interest on account of their excellent properties and potential applicable importance in many fields. Particularly, we recently found that poly(thymine) (poly T) could template the formation of fluorescent copper nanoparticles (CuNPs), offering admirable potential as novel functional biochemical probes. However, exploration of poly T-templated CuNPs for application is still at a very early stage. We report herein for the first example to develop a novel ultrasensitive label-free method for the nuclease (S1 nuclease as a model system) assay, and its inhibitors screening using the poly T-templated fluorescent CuNPs. In this assay, the signal reporter of poly T of 30 mer (T30) kept the original long state in the absence of nuclease, which could effectively template the formation of fluorescent CuNPs. In the presence of nuclease, poly T was digested to mono- or oligonucleotide fragments with decrease of fluorescence. The proposed method was low-cost and simple in its operation without requirement for complex labeling of probe DNA or sophisticated synthesis of the fluorescent compound. The assay process was very rapid with only 5 min for the formation of fluorescent CuNPs. The capabilities for target detection from complex fluids and screening of nuclease inhibitors were verified. A high sensitivity exhibited with a detectable minimum concentration of 5 × 10(-7) units μL(-1) S1 nuclease, which was about 1-4 orders of magnitude more sensitive than the developed approaches.

Poly(thymine)-Templated Copper Nanoparticles as a Fluorescent Indicator for Hydrogen Peroxide and Oxidase-Based Biosensing

Biomineralized fluorescent metal nanoparticles have attracted considerable interest in many fields by virtue of their excellent properties in synthesis and application. Poly(thymine)-templated fluorescent copper nanoparticles (T-CuNPs) as a promising nanomaterial has been exploited by us recently and displays great potential for signal transducing in biochemical analysis. However, the application of T-CuNPs is rare and still at an early stage. Here, a new fluorescent analytical strategy has been developed for H2O2 and oxidase-based biosensing by exploiting T-CuNPs as an effective signal indicator. The mechanism is mainly based on the poly(thymine) length-dependent formation of T-CuNPs and the probes oxidative cleavage. In this assay, the probe T40 can effectively template the formation of T-CuNPs by a fast in situ manner in the absence of H2O2, with high fluorescent signal, while the probe is cleaved into short-oligonucleotide fragments by hydroxyl radical (·OH) which is formed from the Fenton reaction in the presence of H2O2, leading to the decline of fluorescence intensity. By taking advantage of H2O2 as a mediator, this strategy is further exploited for oxidase-based biosensing. As the proof-of-concept, glucose in human serum has been chosen as the model system and has been detected, and its practical applicability has been investigated by assay of real clinical blood samples. Results demonstrate that the proposed strategy has not only good detection capability but also eminent detection performance, such as simplicity and low-cost, holding great potential for constructing effective sensors for biochemical and clinical applications.

Target-Catalyzed Dynamic Assembly-Based Pyrene Excimer Switching for Enzyme-Free Nucleic Acid Amplified Detection

Because of the intrinsic importance of nucleic acid as biotargets, the simple and sensitive detection of nucleic acid is very essential for biological studies and medical diagnostics. Herein, a new strategy for enzyme-free nucleic acid amplified detection has been opened up by combining the signal-amplification capability of target-catalyzed dynamic assembly with the spatially sensitive fluorescent signal of the pyrene excimer. In this strategy, three metastable pyrene-labeled hairpin DNA probes were designed as assembly components, which were kinetically handicapped from cross-opening in the absence of the target DNA. However, in the presence of the target, the dynamic assembly of branched junctions was circularly catalyzed and accompanied by the switching of the pyrene excimer which emits at ~488 nm. Thus, the target DNA could be detected by this simple mix-and-detect amplification method, without expensive and perishable protein enzymes. A good detection capability exhibited with a detectable minimum target concentration of 10 pM, which was comparable to or even better than some reported enzyme-dependent amplification methods, and the potential for the target detection from complex fluids was verified. In addition, as a novel transformation of dynamic DNA assembly technology into enzyme-free signal-amplification analytical application, we infer that the proposed strategy will hold promising potential for application in a wider range of fields, including aptamer-based non-nucleic acid target sensing, biomedicine, and bioimaging.

Visual and portable strategy for copper(II) detection based on a striplike poly(thymine)-caged and microwell-printed hydrogel.

Due to its importance to develop strategies for copper(II) (Cu(2+)) detection, we here report a visual and portable strategy for Cu(2+) detection based on designing and using a strip-like hydrogel. The hydrogel is functionalized through caging poly(thymine) as probes, which can effectively template the formation of fluorescent copper nanoparticles (CuNPs) in the presence of the reductant (ascorbate) and Cu(2+). On the hydrogels surface, uniform wells of microliter volume (microwells) are printed for sample-injection. When the injected sample is stained by Cu(2+), fluorescent CuNPs will be in situ templated by poly T in the hydrogel. With ultraviolet (UV) irradiation, the red fluorescence of CuNPs can be observed by naked-eye and recorded by a common camera without complicated instruments. Thus, the strategy integrates sample-injection, reaction and indication with fast signal response, providing an add-and-read manner for visual and portable detection of Cu(2+), as well as a strip-like strategy. Detection ability with a detectable minimum concentration of 20 μM and practically applicable properties have been demonstrated, such as resistance to environmental interference and good constancy, indicating that the strategy holds great potential and significance for popular detection of Cu(2+), especially in remote regions. We believe that the strip-like hydrogel-based methodology is also applicable to other targets by virtue of altering probes.

dsDNA-templated fluorescent copper nanoparticles: poly(AT-TA)-dependent formation

In this work, poly(AT-TA) is found as the specific sequence composition which contributes to the formation of dsDNA-templated fluorescent copper nanoparticles. The finding will be helpful in wide fields, such as constructing DNA-templated nanodevices and designing biochemical nano-probes.

Ligation-rolling circle amplification combined with γ-cyclodextrin mediated stemless molecular beacon for sensitive and specific genotyping of single-nucleotide polymorphism.

A novel approach for highly sensitive and selective genotyping of single-nucleotide polymorphism (SNP) has been developed based on ligation-rolling circle amplification (L-RCA) and stemless molecular beacon. In this approach, two tailored DNA probes were involved. The stemless molecular beacon, formed through the inclusion interactions of γ-cyclodextrin (γ-CD) and bis-pyrene labeled DNA fragment, was served as signal probe. In the absence of mutant target, the two pyrene molecules were bound in the γ-CD cavity to form an excimer and showed a strong fluorescence at 475 nm. It was here named γ-CD-P-MB. The padlock DNA probe was designed as recognition probe. Upon the recognition of a point mutation DNA targets, the padlock probe was ligated to generate a circular template. An RCA amplification was then initiated using the circular template in the presence of Phi29 polymerase and dNTPs. The L-RCA products, containing repetitive sequence units, subsequently hybridized with the γ-CD-P-MB. This made pyrene molecules away from γ-CD cavity and caused a decrease of excimer fluorescence. As a proof-of-concept, SNP typing of β-thalassemia gene at position -28 was investigated using this approach. The detection limit of mutated target was determined to be 40 fM. In addition, DNA ligase offered high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant target even when the ratio of the wildtype to the mutant is 999:1. Given these attractive characteristics, the developed approach might provide a great genotyping platform for pathogenic diagnosis and genetic analysis.

Dumbbell DNA-templated CuNPs as a nano-fluorescent probe for detection of enzymes involved in ligase-mediated DNA repair

DNA repair processes are responsible for maintaining genome stability. Ligase and polynucleotide kinase (PNK) have important roles in ligase-mediated DNA repair. The development of analytical methods to monitor these enzymes involved in DNA repair pathways is of great interest in biochemistry and biotechnology. In this work, we reported a new strategy for label-free monitoring PNK and ligase activity by using dumbbell-shaped DNA templated copper nanoparticles (CuNPs). In the presence of PNK and ligase, the dumbbell-shaped DNA probe (DP) was locked and could resist the digestion of exonucleases and then served as an efficient template for synthesizing fluorescent CuNPs. However, in the absence of ligase or PNK, the nicked DP could be digested by exonucleases and failed to template fluorescent CuNPs. Therefore, the fluorescence changes of CuNPs could be used to evaluate these enzymes activity. Under the optimal conditions, highly sensitive detection of ligase activity of about 1U/mL and PNK activity down to 0.05U/mL is achieved. To challenge the practical application capability of this strategy, the detection of analyte in dilute cells extracts was also investigated and showed similar linear relationships. In addition to ligase and PNK, this sensing strategy was also extended to the detection of phosphatase, which illustrates the versatility of this strategy.

Nucleic acid tool enzymes-aided signal amplification strategy for biochemical analysis: status and challenges

AbstractOwing to their highly efficient catalytic effects and substrate specificity, the nucleic acid tool enzymes are applied as ‘nano-tools’ for manipulating different nucleic acid substrates both in the test-tube and in living organisms. In addition to the function as molecular scissors and molecular glue in genetic engineering, the application of nucleic acid tool enzymes in biochemical analysis has also been extensively developed in the past few decades. Used as amplifying labels for biorecognition events, the nucleic acid tool enzymes are mainly applied in nucleic acids amplification sensing, as well as the amplification sensing of biorelated variations of nucleic acids. With the introduction of aptamers, which can bind different target molecules, the nucleic acid tool enzymes-aided signal amplification strategies can also be used to sense non-nucleic targets (e.g., ions, small molecules, proteins, and cells). This review describes and discusses the amplification strategies of nucleic acid tool enzymes-aided biosensors for biochemical analysis applications. Various analytes, including nucleic acids, ions, small molecules, proteins, and cells, are reviewed briefly. This work also addresses the future trends and outlooks for signal amplification in nucleic acid tool enzymes-aided biosensors. Graphical abstractNucleic acid tool enzymes-aided signal amplification sensing

Oligonucleotide-templated rapid formation of fluorescent gold nanoclusters and its application for Hg2+ ions sensing

In this work, we developed a simple, rapid and mild strategy for synthesis of DNA-templated fluorescent gold nanoclusters (AuNCs) through association of gold ions to DNA templates and reduction with 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). After systematical investigation on the formation of fluorescent AuNCs by using different DNA templates, C5 DNA was found as the best template for the formation of fluorescent AuNCs in this system. This process could be completed within 5min after the reaction beginning under ambient conditions. The prepared C5-AuNCs displayed good blue emission and photostability. Furthermore, this C5-AuNCs could be applied to detecting Hg2+ ions specifically based on the specific and strong interaction between Hg2+ and Au+. The C5-AuNCs provided excellent selectivity for Hg2+ ions over other metal ions, and also high sensitivity, with a detection limit of 50nM. As biocompatible, environmentally-friendly, and synthesis-rapid, this C5-AuNCs probe appears to be promising candidate for biochemical sensing via simple modification of template DNA.

Triple-helix molecular switch-induced hybridization chain reaction amplification for developing a universal and sensitive electrochemical aptasensor

In this work, a universal and sensitive “signal-on” electrochemical aptasensor platform has been developed based on a triple-helix molecular switch (THMS)-induced hybridization chain reaction (HCR) amplification. This aptasensor platform system consists of a THMS-based molecular recognition process in a homogeneous solution and a HCR amplification on a gold electrode. In the absence of a target, the aptamer sequence is flanked by two arm segments (APT) and the triplex-forming oligonucleotide (TFO), forming a rigid THMS. It is in the eT off state. However, upon the introduction of a target, the interaction between the target and the APT leads to the dissociation of the THMS and thereby liberates the TFO, allowing the TFO to hybridize with the capture probe (CP) DNA and trigger the formation of dsDNA polymers through in situ HCR amplification. The dsDNA polymers cause the electrostatic attraction of numerous electroactive indicators [Ru(NH3)6]3+, resulting in significantly amplified electrochemical signal output. It is in the eT on state. As proof-of-principle, we use this approach to detect adenosine and human α-thrombin (Tmb), achieving lowest limit of detection values of 0.6 nM and 70.9 pM, respectively. As an electrochemical aptasensor platform, its universality can be easily realized by altering only the sequence of the APT, which provides a promising alternative to the electrochemical detection of a variety of analytes and may have potential applications in biomedical research and clinical diagnosis.