Dinggeng He

Concatemeric dsDNA-templated copper nanoparticles strategy with improved sensitivity and stability based on rolling circle replication and its application in microRNA detection.

DNA-templated copper nanoparticles (CuNPs) have emerged as promising fluorescent probes for biochemical assays, but the reported monomeric CuNPs remain problematic because of weak fluorescence and poor stability. To solve this problem, a novel concatemeric dsDNA-templated CuNPs (dsDNA-CuNPs) strategy was proposed by introducing the rolling circle replication (RCR) technique into CuNPs synthesis. In this strategy, a short oligonucleotide primer could trigger RCR and be further converted to a long concatemeric dsDNA scaffold through hybridization. After the addition of copper ions and ascorbate, concatemeric dsDNA-CuNPs could effectively form and emit intense fluorescence in the range of 500-650 nm under a 340 nm excitation. In comparison with monomeric dsDNA-CuNPs, the sensitivity of concatemeric dsDNA-CuNPs was greatly improved with ~10,000 folds amplification. And their fluorescence signal was detected to reserve ~60% at 2.5 h after formation, revealing ~2 times enhanced stability. On the basis of these advantages, microRNA let-7d was selected as the model target to testify this strategy as a versatile assay platform. By directly using let-7d as the primer in RCR, the simple, low-cost, and selective microRNA detection was successfully achieved with a good linearity between 10 and 400 pM and a detection limit of 10 pM. The concatemeric dsDNA-CuNPs strategy might be widely adapted to various analytes that can directly or indirectly induce RCR.

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.

A Light-Responsive Reversible Molecule-Gated System Using Thymine-Modified Mesoporous Silica Nanoparticles

In this paper, a reversible light-responsive molecule-gated system based on mesoporous silica nanoparticles (MSN) functionalized with thymine derivatives is designed and demonstrated. The closing/opening protocol and release of the entrapped guest molecules is related by a photodimerization-cleavage cycle of thymine upon different irradiation. In the system, thymine derivatives with hydrophilicity and biocompatibility were grafted on the pore outlets of MSN. The irradiation with 365 nm wavelength UV light to thymine-functionalized MSN led to the formation of cyclobutane dimer in the pore outlet, subsequently resulting in blockage of pores and strongly inhibiting the diffusion of guest molecules from pores. With 240 nm wavelength UV light irradiation, the photocleavage of cyclobutane dimer opened the pore and allowed the release of the entrapped guest molecules. As a proof-of-the-concept, Ru(bipy)(3)(2+) was selected as the guest molecule. Then the light-responsive loading and release of Ru(bipy)(3)(2+) were investigated. The results indicated that the system had an excellent loading amount (53 μmol g(-1) MSN) and controlled release behavior (82% release after irradiation for 24 h), and the light-responsive loading and release procedure exhibited a good reversibility. Besides, the light-responsive system loaded with Ru(bipy)(3)(2+) molecule could also be used as a light-switchable oxygen sensor.

A pH-responsive polymer/mesoporous silica nano-container linked through an acid cleavable linker for intracellular controlled release and tumor therapy in vivo†

This paper proposes a pH-responsive polymer/mesoporous silica nano-container linked through an acid cleavable linker for intracellular controlled release and tumor therapy in vivo. In this system, the poly(acrylic acid) homopolymer (PAA), as a nanoscopic cap, is grafted onto the MSN through an acid cleavable linker (PAA-ACL-MSN). Doxorubicin (DOX), as a model drug, is used to assess the drug release behaviors and tumor therapy. At neutral pH, the linker is intact, resulting in blockage of pores and package of DOX. By the degradation of the linker at acidic pH, the grafted PAA is removed, which gives rise to uncapping and the subsequent pH-responsive controlled release of DOX. In vitro studies using a nasopharyngeal carcinoma cell line (HNE-1) prove that DOX loaded PAA-ACL-MSN (DOX@PAA-ACL-MSN) is endocytosed and demonstrates efficient operation at lysosomal pH, leading to significant cytotoxicity. As a preliminary tumor therapy in vivo, the progressive tumor development and inhibition following DOX@PAA-ACL-MSN treatment is monitored using bioluminescence imaging. By the examination of cell proliferation in tumor tissues and the comparison of body weight, it is revealed that the DOX@PAA-ACL-MSN is superior to free DOX in terms of therapeutic efficacy and side effects due to the enhanced permeability and retention effects and lower pH in tumor areas. We believe that this developed MSN based delivery system will provide a promising nanodevice for tumor therapy.

Natural Gelatin Capped Mesoporous Silica Nanoparticles for Intracellular Acid-Triggered Drug Delivery

This paper proposed a natural gelatin capped mesoporous silica nanoparticles (MSN@Gelatin) based pH-responsive delivery system for intracellular anticancer drug controlled release. In this system, the gelatin, a proteinaceous biopolymer derived from the processing of animal collagen, was grafted onto the MSN to form a capping layer via temperature-induced gelation and subsequent glutaraldehyde mediated cross-linking, resulting in gelatin coated MSN. At neutral pH, the gelatin capping layer could effectively prohibit the release of loaded drug molecules. However, the slightly acidic environment would lead to enhanced electrostatic repulsion between the gelatin and MSN, giving rise to uncapping and the subsequent controlled release of the entrapped drug. As a proof-of-concept, doxorubicin (DOX) was selected as the model anticancer drug. The loading and pH-responsive release experiments demonstrated that the system had excellent loading efficiency (47.3 mmol g(-1) SiO2), and almost no DOX was leaked at neutral. After being in the slightly acidic condition, the DOX release from the DOX-loaded MSN@Gelatin (DOX/MSN@Gelatin) occurred immediately. The cellular uptake and release studies using Hep-G2 hepatoma cells indicated that the DOX/MSN@Gelatin could be endocytosed and accumulated within lysosomes. Triggered by acidic endosomal pH, the intracellular release of the loaded DOX was obviously eventuated. Further cell viability results demonstrated that DOX/MSN@Gelatin exhibited dose-dependent toxicity and high killing efficacy (IC50 = 17.27 ± 0.63 μg mL(-1)), whereas the MSN@Gelatin showed negligible cytotoxicity (IC50 > 100 μg mL(-1)). This biocompatible and effective delivery system will provide great potential for developing delivery of cancer therapeutic agents.

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.

Regenerable multifunctional mesoporous silica nanocomposites for simultaneous detection and removal of mercury(II).

Mercury (Hg(2+)) is a highly toxic and widespread environmental pollutant. Herein, a regenerable and highly selective core-shell structured magnetic mesoporous silica nanocomposite with functionalization of thymine (T) and T-rich DNA (denoted as Fe3O4@nSiO2@mSiO2-T-TRDNA nanocomposite) has been developed for simultaneous detection and removal of Hg(2+). In this work, the thymine and T-rich DNA were immobilized onto the interior and exterior surface of outermost mesoporous silica, respectively. The detection mechanism is based on Hg(2+)-mediated hairpin structure formed by T-rich DNA functionalized on the exterior surface of the nanocomposites, where, upon addition of SYBR Green I dye, strong fluorescence is observed. In the absence of Hg(2+), however, addition of the dye results in low fluorescence. The limit of detection for Hg(2+) in a buffer is 2 nM by fluorescence spectroscopy. Simultaneously, the Fe3O4@nSiO2@mSiO2-T-TRDNA nanocomposite features a selective binding with Hg(2+) between two thymines immobilized at the interior surface of the mesopores and exhibits efficient and convenient Hg(2+) removal by a magnet. Kinetic study reveals that the Hg(2+) removal is a rapid process with over 80% of Hg(2+) removed within approximately 1 h. The applicability of the developed nanocomposites is demonstrated to detect and remove Hg(2+) from samples of Xiangjiang river water spiked with Hg(2+). In addition, distinguishing aspects of the Fe3O4@nSiO2@mSiO2-T-TRDNA nanocomposites for Hg(2+) detection and removal also include the regeneration using a simple acid treatment and resistance to nuclease digestion. Similar process can be used to functionalize the Fe3O4@nSiO2@mSiO2 nanocomposites with other nucleic acids and small molecules for environmental and biomedical applications.

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.

Remote-controlled drug release from graphene oxide-capped mesoporous silica to cancer cells by photoinduced pH-jump activation.

Remote light control of drug release enhances our ability to address the complexity of biological systems because of its remarkable spatial/temporal resolution. Here, a new class of remote-controlled release system by incorporating photoacid generator (PAG) into graphene oxide-capped mesoporous silica was designed for delivering drug payloads to cancer cells via photoinduced pH-jump activation. PAG was immobilized on pore wall of the boronic acid-grafted mesoporous silica via strong physical adsorption, and then the nanoparticle was capped with graphene oxide sheet by an acid-labile boroester bond, leading to the formation of nanogated ensemble (MSP-BA-GO). Illuminating with a UV light, PAG generated a pH jump, which induced cleavage of the boroester linkers and thus resulted in the uncapping of pore gates. Moreover, folic acid-modified, doxorubicin (DOX)-loaded MSP-BA-GO (DOX@MSP-BA-GOF) showed selective cell internalization via receptor-mediated endocytosis and subsequent released DOX by the remote illumination. We envisioned that this remote-controlled drug delivery system could find potential applications for cancer therapy.