Fe-doped carbon dots: a novel fluorescent nanoprobe for cellular hypochlorous acid imaging.

Real-time monitoring of hypochlorous acid (HClO) in biological systems is of great significance for exploring and regulating its pathological functions because abnormal production of HClO is closely related with many diseases, such as atherosclerosis, rheumatoid arthritis, and cancer. Herein, we developed a reliable fluorescent Fe-doped carbon dots (Fe-CDs) for the sensitive and selective detection of biological HClO using ferrocenecarboxylic acid and m-phenylenediamine as precursors through a one-step solvothermal procedure. The Fe-CDs exhibited excellent a wide HClO detection range from 20 nmol/L to 1000 nmol/L with corresponding limits of detection at 7.8 nmol/L. The sensing mechanism is based on the chemical oxidation of the hydroxyl groups on the surface of Fe-CDs by HClO. In addition, Fe-CDs also displayed high photoluminescence yield, excitation-independence emission, as well as good biocompatibility, enabling the successful imaging of endogenous and exogenous HClO in HeLa cells. These results revealed that Fe-CDs holds great promise as a robust fluorescent probe for investigating HClO-mediated biological events.

Simultaneous Response of Mitochondrial MicroRNAs to Identify Cell Apoptosis with Multiple Responsive Intelligent DNA Biocomputing Nanodevices.

Challenges remained in precisely real-time monitoring of apoptotic molecular events at the subcellular level. Herein, we developed a new type of intelligent DNA biocomputing nanodevices (iDBNs) that responded to mitochondrial microRNA-21 (miR-21) and microRNA-10b (miR-10b) simultaneously which were produced during cell apoptosis. By hybridizing two hairpins (H1 and H2) onto DNA nanospheres (DNSs) that had been previously modified with mitochondria-targeted triphenylphosphine (TPP) motifs, iDBNs were assembled in which two localized catalytic hairpins self-assembly (CHA) reactions occurred upon the co-stimulation of mitochondrial miR-21 and miR-10b to perform AND logic operations, outputting fluorescence resonance energy transfer (FRET) signals for sensitive intracellular imaging during cell apoptosis. Owing to the spatial confinement effects of DNSs, it was discovered that iDBNs had a high efficiency and speed of logic operations by high local concentrations of H1 and H2, making the simultaneous real-time responses of mitochondrial miR-21 and miR-10b reliable and sensitive during cell apoptosis. These results demonstrated that iDBNs were simultaneously responsive to multiple biomarkers, which greatly improved the detection accuracy to identify the cell apoptosis, demonstrating that iDBNs are highly effective and reliable for the diagnosis of major disease and screening of anticancer drugs.

A high-integrated DNA biocomputing platform for MicroRNA sensing in living cells.

DNA logic computing has captured increasing interest due to its ability to assemble programmable DNA computing elements for disease diagnosis, gene regulation, and targeted therapy. In this work, we developed an aptamer-equipped high-integrated DNA biocomputing platform (HIDBP-A) with a dual-recognition function that enabled cancer cell targeting. Dual microRNAs were the input signals and can perform AND logic operations. Compared to the free DNA biocomputing platform (FDBP), the integration of all computing elements into the same DNA tetrahedron greatly improved logic computing speed and efficiency owing to the confinement effect reflected by the high local concentration of computing elements. As a proof of concept, the utilization of microRNA as the input signal was beneficial for improving the scalability and flexibility of the sequence design of the logic nano-platform. Given that the different microRNAs were over-expressed in cancer cells, this new HIDBP-A has great promise in accurate diagnosis and logic-controlled disease treatment.

Simultaneous Imaging of Dual microRNAs in Cancer Cells through Catalytic Hairpin Assembly on a DNA Tetrahedron.

Accurate detection and imaging of tumor-related microRNA (miRNA) in living cells hold great promise for early cancer diagnosis and prognosis. One of the challenges is to develop methods that enable the identification of multiple miRNAs simultaneously to further improve the detection accuracy. Herein, a simultaneous detection and imaging method of two miRNAs was established by using a programmable designed DNA tetrahedron nanostructure (DTN) probe that includes a nucleolin aptamer (AS1411), two miRNA capture strands, and two pairs of metastable catalytic hairpins at different vertexes. The DTN probe exhibited enhanced tumor cell recognition ability, excellent stability and biocompatibility, and fast miRNA recognition and reaction kinetics. It was found that the DTN probe could specifically enter tumor cells, in which the capture strand could hybridize with miRNAs and initiate the catalytic hairpin assembly (CHA) only when the overexpressed miR-21 and miR-155 existed simultaneously, resulting in a distinct fluorescence resonance energy transfer signal and demonstrating the feasibility of this method for tumor diagnosis.

Hierarchical Hybridization Chain Reaction for Amplified Signal Output and Cascade DNA Logic Circuits.

In this work, we propose a three-layer hierarchical hybridization chain reaction (3L hHCR) composed of 1stHCR, 2ndHCR, and 3rdHCR to achieve robust signal amplification efficiency and broaden the applied range of HCR-based systems. In principle, the execution of superior HCR generates the formation of the initiator (named as 2ndI or 3rdI) of the subordinate HCR that relies on the introduction of the target sequence (1stI). To avoid the high background signal of the 3L hHCR system, a strategy of "splitting reconstruction" was adopted. The initiator of the subordinate HCR was designed as two separate fragments (splitting) that are obtained together (reconstruction) for the motivation of the subordinate HCR after the completion of the superior HCR. The implementation of the entire 3L hHCR system generates significant fluorescence recovery that derives from the impediment of Förster resonance energy transfer between fluorophore and quencher; thus, ultrasensitive detection of 1stI in the range of 50 pM to 10 nM can be achieved. Surprisingly, when the concentration of 1stI is lower than 1 nM, the 3L hHCR shows excellent ability to discriminate against various concentrations of 1stI, which is better than that of the 2L hHCR I system. Due to the hierarchical self-assembly mechanism, the 3L hHCR can also be reliably operated as a cascade AND logic gate with a high specificity and molecular keypad lock with a prompt error-reporting function. Furthermore, the 3L hHCR-based molecular keypad lock also shows potential application in the accurate diagnosis of cancer. The 3 L hHCR shows visionary prospects in biosensing and the fabrication of advanced biocomputing networks.

Nucleolin-Targeted DNA Nanotube for Precise Cancer Therapy through Förster Resonance Energy Transfer-Indicated Telomerase Responsiveness.

Precise drug delivery holds great promise in cancer treatment but still faces challenges in controllable drug release in tumor cells specifically. Herein, a nucleolin-targeted and telomerase-responsive DNA nanotube for drug release was developed. First, a DNA nanosheet with four capture strands on its surface was prepared, which could bind and load ricin A chain (RTA). The RTA-loaded nanosheet was further converted into a DNA nanotube with a high Förster resonance energy transfer (FRET) efficiency in the presence of a Cy3-modified DNA fastener by hybridizing with the Cy5-modified DNA and another DNA-containing telomerase primer sequence along the long sides. Moreover, the aptamer of nucleolin was assembled on the DNA nanotube by combining with the hybrid chain at the terminal. The aptamer-functionalized and RTA-loaded DNA nanotube displayed enhanced tumor permeability and precise drug release in response to the telomerase in tumor cells, following the change of the FRET signal and RTA-induced cell death. Moreover, the DNA nanotube was applied successfully in vivo, and there was an obvious inhibition of tumor growth on xenograft-bearing mice following systemic administration, indicating that the constructed DNA nanotube represents a promising platform for precise RTA delivery in target cancer therapy.

DNA nanosheet as an excellent fluorescence anisotropy amplification platform for accurate and sensitive biosensing.

Recently, various inorganic nanomaterials have been used as fluorescence anisotropy (FA) enhancers for biosensing successfully. However, most of them are size-uncontrollable and possess an intensive fluorescence quenching ability, which will seriously reduce the accuracy and sensitivity of FA method. Herein, we report a two-dimensional DNA nanosheet (DNS) without fluorescence quenching effect as a novel FA amplification platform. In our strategy, fluorophore-labeled probe DNA (pDNA) is linked onto the DNS surface through the hybridization with the handle DNA (hDNA) that extended from the DNS, resulting in the significantly enhanced FA value. After the addition of target, the pDNA was released from the DNS surface due to the high affinity between the hDNA and target, and the FA was decreased. Thus, target could be detected by the significantly decreased FA value. The linear range was 10-50 nM and the limit of detection was 8 nM for the single-stranded DNA detection. This new method is general and has been also successfully applied for the detection of ATP and thrombin sensitively. Our method improved the accuracy of FA assay and has great potential to detect series of biological analytes in complex biosensing systems.

Silver nanoparticles deposited on graphene oxide for ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarker.

Graphene oxide (GO) exhibits distinctive Raman scattering features for its high frequency D (disordered) and tangential modes (G-band), which are characteristically sharp at 1580 cm-1 and 1350 cm-1, respectively, but are too weak for sensitive quantitation purposes. By depositing silver nanoparticles on the surface of GO in this contribution, both D and G bands of GO become enhanced. The enzyme label of this method controls the dissolution of silver nanoparticles on the surface of GO through hydrogen peroxide which is produced by the oxidation of the enzyme substrate. With the dissolution of the silver nanoparticles a greatly decreased SERS signal of GO was obtained. This strategy involves dual signal amplification of the enzyme and nanocomposites to improve the detection sensitivity. As a proof of concept, prostate specific antigen (PSA), a biomarker for prostate cancer, is successfully detected as a target by forming a sandwich structure in immunoassay. The SERS immunoassay possesses excellent analytical performance in the range 0.5 pg mL-1 to 500 pg mL-1 with a limit of detection of 0.23 pg mL-1, making the detection of PSA serum samples from prostate cancer patients satisfactory, demonstrating that the sensitive enzyme-assisted dissolved AgNPs SERS immunoassay of PSA has potential applications in clinical diagnosis.

Reduced graphene oxide coated Cu2-xSe nanoparticles for targeted chemo-photothermal therapy.

Recently, copper chalcogenide semiconductors have been reported as new near-infrared (NIR) photothermal agents. However, it is difficult to modify them with recognition molecules, and their photothermal conversion efficiencies are relatively low, making it difficult to achieve the targeted photothermal ablation of cancer cells with a high efficiency. In this study, reduced graphene oxide (rGO) was first coated on the surface of Cu2-xSe nanoparticles (NPs) to provide abundant functional groups for the next modification and to increase the photothermal conversion efficiency. Then, doxorubicin (DOX) was loaded and folic acid (FA) molecules were covalently linked onto the surface of Cu2-xSe/rGO nanocomposites. The formed DOX@Cu2-xSe@rGO-FA nanocomposites were successfully used as chemo-photothermal agents for the targeted killing of cancer cells by utilizing the recognition ability of FA, chemotherapy effect of DOX and photothermal effects of rGO and Cu2-xSe NPs. Under the 980-nm NIR laser irradiation, the nanocomposites showed significantly enhanced chemo-photothermal therapy effect, which can be potentially applied in the nanomedicine field.

Metal-organic gel enhanced fluorescence anisotropy for sensitive detection of prostate specific antigen.

In this contribution, we demonstrated that Cu-based metal-organic gel (Cu-MOG) was able to serve as a novel amplification platform for fluorescence anisotropy (FA) assay for the first time, which was confirmed by the sensitive detection of a common cancer biomarker, prostate specific antigen (PSA). The dye-labeled probe aptamer (PA) product was adsorbed onto the benzimidazole derivative-containing Cu-MOG via electrostatic incorporation and strong π-π stacking interactions, which significantly increased the FA value due to the enlargement of the molecular volume of the PA/Cu-MOG complex. With the introduction of target PSA, the FA value was obviously decreased on account of the specific recognition between PSA and PA which resulted in the detachment of PA from the surface of MOG. The linear range was from 0.5-8ng/mL, with a detection limit of 0.33ng/mL. Our work has thus helped to demonstrate promising application of MOG material in the fields of biomolecules analysis and disease diagnosis.

An Enzyme-Free DNA Circuit-Assisted Graphene Oxide Enhanced Fluorescence Anisotropy Assay for MicroRNA Detection with Improved Sensitivity and Selectivity.

Graphene oxide (GO) has been proven as an outstanding fluorescence anisotropy (FA) amplifier. Yet the traditional GO amplified FA assays lack high sensitivity because of the 1:1 binding ratio between target and dye-modified probe. Herein, we report a new target-catalyzed hairpin assembly (CHA), an enzyme-free DNA circuit, assisted GO amplified FA strategy for microRNA-21 (miRNA-21) detection. In the presence of miRNA-21, the CHA was initiated and plenty of H1-H2 duplexes were produced continuously. The obtained H1-H2 duplex could induce the formation of a H1-H2-probe DNA (pDNA) complex by the toehold-mediated strand exchange reaction, which led the dye-modified pDNA to leave away from the GO surface, resulting in a decreased FA of the system. By monitoring the decrease of FA, miRNA-21 could be detected in the range of 0-16 nM. The limit of detection (LOD, 3σ) was 47 pM, which was 194 times lower than that without CHA. In addition, the selectivity of this method has also been enhanced greatly as compared to that without CHA. Our method has great potential to be applied for detecting different types of targets and monitoring diverse molecular interactions by adapting the corresponding nucleotide sequence.

A dynamic cell entry pathway of respiratory syncytial virus revealed by tracking the quantum dot-labeled single virus.

Studying the cell entry pathway at the single-particle level can provide detailed and quantitative information for the dynamic events involved in virus entry. Indeed, the viral entry dynamics cannot be monitored by static staining methods used in cell biology, and thus virus dynamic tracking could be useful in the development of effective antiviral strategies. Therefore, the aim of this work was to use a quantum dot-based single-particle tracking approach to monitor the cell entry behavior of the respiratory syncytial virus (RSV) in living cells. The time-lapse fluorescence imaging and trajectory analysis of the quantum dot-labeled RSV showed that RSV entry into HEp-2 cells consisted of a typical endocytosis trafficking process. Three critical events during RSV entry were observed according to entry dynamic and fluorescence colocalization analysis. Firstly, RSV was attached to lipid rafts of the cell membrane, and then it was efficiently delivered into the perinuclear region within 2 h post-infection, mostly moving and residing into the lysosome compartment. Moreover, the relatively slow velocity of RSV transport across the cytoplasm and the formation of the actin tail indicated actin-based RSV motility, which was also confirmed by the effects of cytoskeletal inhibitors. Taken together, these findings provided new insights into the RSV entry mechanism and virus-cell interactions in RSV infection that could be beneficial in the development of antiviral drugs and vaccines.

An enzyme-induced Au@Ag core-shell nanoStructure used for an ultrasensitive surface-enhanced Raman scattering immunoassay of cancer biomarkers.

Early detection of cancer is helpful for the control and prevention of diseases. Due to the low content of cancer biomarkers in the early disease phases, however, an ultrasensitive and selective method is critical. In this contribution, an ultrasensitive surface-enhanced Raman scattering (SERS) immunoassay is newly developed with the principle of introducing a common enzyme-induced deposition (EID) reaction to coat a silver layer on the surface of gold nanoparticles and to form a core-shell nanostructure of Au@Ag. By using alkaline phosphatase (ALP) to dephosphorylate its substrate, 2-phospho-l-ascorbic acid trisodium salt (AAP), to form vitamin C, silver ions could be reduced into silver atoms and coated on the surface of the AuNPs; a greatly enhanced SERS signal was then obtained. As a proof of concept, α-fetoprotein (AFP) was detected as a target, which is a biomarker of liver cancer. Excellent analytical performance of the SERS immunoassay could be achieved in the range from 0.5 to 100 pg mL-1 with a limit of detection of 0.081 pg mL-1 (3σ). Identical results could be obtained by using the newly proposed SERS immunoassay for the clinical detection of AFP in serum samples of patients to those clinically obtained by chemiluminescence immunoassays, demonstrating the potential applications of the developed method in clinical diagnosis.

His-tag based in situ labelling of progeny viruses for real-time single virus tracking in living cells.

Tracking virus infection events in live cells is useful for understanding the mechanism of virus infection, and fluorescent labelling is a critical step. Herein a noninvasive strategy for labelling viruses with His-tags was developed by in situ modifying the cell surface proteins with polypeptides containing His-tags during progeny virus assembly. The His-tagged viruses were further conjugated with Ni2+-nitrilotriacetate complex modified quantum dots, and retained their infectivity for real-time single virus tracking in living cells.

A sensitive surface-enhanced Raman scattering enzyme-catalyzed immunoassay of respiratory syncytial virus.

Respiratory viruses have become a major global health challenge which would benefit from advances in screening methods for early diagnosis. Respiratory syncytial virus (RSV) is one of the most important pathogen causing severe lower respiratory tract infections. Here we present a novel surface-enhanced Raman scattering (SERS) enzyme-catalyzed immunoassay of RSV by employing peroxidase substrate 3, 3'-5, 5'-tetramethylbenzidine (TMB) as Raman molecule. Horseradish peroxidase (HRP) attached to the detection antibody in a novel sandwich immunoassay catalyzes the oxidation of TMB by H2O2 to give a radical cation (TMB(+)), which could be easily adsorbed on the negatively charged surface of silver nanoparticles (AgNPs) through electrostatic interaction, inducing the aggregation of AgNPs and thus giving a strong SERS signal. A linear relationship was obtained between the Raman intensity and the amount of RSV in the range from 0.5 to 20 pg/mL, and the minimum detectable concentration of this SERS-based enzyme immunoassay was 0.05 pg/mL, which was 20 times lower than that found in the colorimetric method.

DNA-AuNP networks on cell membranes as a protective barrier to inhibit viral attachment, entry and budding.

Viral infections have caused numerous diseases and deaths worldwide. Due to the emergence of new viruses and frequent virus variation, conventional antiviral strategies that directly target viral or cellular proteins are limited because of the specificity, drug resistance and rapid clearance from the human body. Therefore, developing safe and potent antiviral agents with activity against viral infection at multiple points in the viral life cycle remains a major challenge. In this report, we propose a new modality to inhibit viral infection by fabricating DNA conjugated gold nanoparticle (DNA-AuNP) networks on cell membranes as a protective barrier. The DNA-AuNPs networks were found, via a plaque formation assay and viral titers, to have potent antiviral ability and protect host cells from human respiratory syncytial virus (RSV). Confocal immunofluorescence image analysis showed 80 ± 3.8% of viral attachment, 91.1 ± 0.9% of viral entry and 87.9 ± 2.8% of viral budding were inhibited by the DNA-AuNP networks, which were further confirmed by real-time fluorescence imaging of the RSV infection process. The antiviral activity of the networks may be attributed to steric effects, the disruption of membrane glycoproteins and limited fusion of cell membrane bilayers, all of which play important roles in viral infection. Therefore, our results suggest that the DNA-AuNP networks have not only prophylactic effects to inhibit virus attachment and entry, but also therapeutic effects to inhibit viral budding and cell-to-cell spread. More importantly, this proof-of-principle study provides a pathway for the development of a universal, broad-spectrum antiviral therapy.

A novel graphene oxide amplified fluorescence anisotropy assay with improved accuracy and sensitivity.

In this contribution, a novel and versatile graphene oxide (GO) amplified fluorescence anisotropy (FA) strategy with improved accuracy and sensitivity for the detection of a panel of molecules, single-stranded DNA (ssDNA), adenosine and thrombin, has been successfully developed.

Nanosilver-based surface-enhanced Raman spectroscopic determination of DNA methyltransferase activity through real-time hybridization chain reaction.

In this manuscript, a nanosilver enhanced SERS strategy was successfully constructed for the determination of DNA methyltransferase activity in soulution combined with hybridization chain reaction (HCR). The proposed method was mainly on the basis of excellent separation ability of magnetic microparticles (MMPs), HCR as signal amplification unit and assembled AgNPs as enhancement substrate. In the presence of M. SssI MTase, the duplex sequence (5'-CCGG-3') tethered to MMPs was methylated, which cannot be cleaved by HpaII endonuclease. The resulted DNA skeleton captured on MMPs then triggered the HCR reaction, generated a polymerized and extended symmetrical sequence, in which more biotin terminal was available for the conjugation of AgNPs-SA, leading to significantly amplified SERS response. When it was used to analyze M. SssI activity, a linear equation ∆ISERS=1215.32+446.80 cM.SssI was obtained with the M. SssI activity ranged from 0.1 to 10.0 U with the correlation coefficient (r(2)) of 0.97. The most important advantage of this method is the combination of SERS and HCR in solution for the first time and its good selectivity, which enabled the detection of even one-base mismatched sequence. The new assay method holds great promising application to be a versatile platform for sensitive, high-throughput detection, and the screening of new anticancer drugs on DNA MTase.

A gold nanoparticles-based colorimetric assay for alkaline phosphatase detection with tunable dynamic range.

In this report, a simple and label-free colorimetric assay was developed for detecting alkaline phosphatase (ALP). Based on the conjugated gold nanoparticle/adenosine triphosphate (AuNP/ATP) sensing system, this assay is highly sensitive and selective. In this system, ATP induces the aggregation of cetyltrimethylammonium bromide (CTAB)-capped AuNPs and ALP stimulates the disaggregation of AuNPs by converting ATP into adenosine through an enzymatic dephosphorylation reaction. Hence, the presence of ALP can be visually observed (gray-to-red color change) and monitored by the shift of the surface plasmon resonance (SPR) absorption band of AuNPs. Furthermore, the dynamic range of the method can be varied by addition of different metal ions (e.g. 100-600unit/L to 5.0-100unit/L and 0.2-20unit/L in the presence of Ca(2+) and Pb(2+), respectively). The feasibility of this sensitive and specific assay with a tunable dynamic range was demonstrated to be consistent even in human serum samples.

A ratiometric fluorescence recognition of guanosine triphosphate on the basis of Zn(II) complex of 1,4-bis(imidazol-1-ylmethyl) benzene.

As one vital member among the family of phosphates, guanosine triphosphate (GTP) not only plays a very important role in many critical biological processes but also closely associates with definite pathological states. Based on the ratiometric fluorescence response of the zinc complex of 1,4-bis(imidazol-1-ylmethyl) benzene (bix) in this contribution, a highly selective recognition of GTP has been successfully developed. The fluorescence of bix-Zn(II) at 289 nm decreased in the presence of GTP with the appearance of one new emission band at 341 nm, resulting in ratiometric fluorescence changes with the concentration of GTP. With that, ratiometric fluorescence recognition for GTP could be effectively established, and so GTP could be successfully discriminated from other structurally similar anions, including ATP and PPi. Furthermore, bix-Zn(II) also has a ratiometric fluorescence response to DNA sequences containing guanine.