A Macrophage Membrane-Coated Cu-WO3-x-Hydro820 Nanoreactor for Treatment and Photoacoustic/Fluorescence Dual-Mode Imaging of Inflamed Liver Tissue.

A disease-targeting nanoplatform that integrates imaging with therapeutic activity would facilitate early diagnosis, treatment, and therapeutic monitoring. To this end, a macrophage membrane-coated Cu-WO3-x-Hydro820 (CWHM) nanoreactor was prepared. This reactor was shown to target inflammatory tissues. The reactive oxygen species (ROS) such as H2O2 and ·OH in inflammatory tissues can react with Hydro820 in the reactor to form the NIR fluorophore IR820. This process allowed photoacoustic/fluorescence dual-mode imaging of H2O2 and ·OH, and it is expected to permit visual diagnosis of inflammatory diseases. The Cu-WO3-x nanoparticles within the nanoreactor shown catalase and superoxide enzyme mimetic activity, allowing the nanoreactor to catalyze the decomposition of H2O2 and ·O2- in inflammatory cells of hepatic tissues in a mouse model of liver injury, thus alleviating the oxidative stress of damaged liver tissue. This nanoreactor illustrates a new strategy for the diagnosis and treatment of hepatitis and inflammatory liver injury.

A dual-core 3D DNA nanomachine based on DNAzyme positive feedback loop for highly sensitive MicroRNA imaging in living cells.

A double 3D DNA walker nanomachine by DNAzyme self-driven positive feedback loop amplification for the detection of miRNA was constructed. This method uses two gold nanoparticles as the reaction core, and because of the spatial confinement effect the local concentration of the reactants increase the collision efficiency was greatly improved. Meanwhile, the introduction of positive feedback loop promotes the conversion efficiency. In presence of miRNA-21, a large amount of DNAzyme was released and hydrolyze the reporter probe, resulting the recovery of fluorescence signal. The linear range for miRNA-21 is 0.5-60 pmol/L, and the detection limit is 0.41 pmol/L (S/N = 3). This nanomachine has been successfully used for accurate detection of miRNA-21 expression levels in cell lysates. At the same time, it can enter cells for intracellular miRNA-21 fluorescence imaging, distinguishing tumor cells from normal cells. This combination of in vitro detection and imaging analysis of living cells can achieve the goal of jointly detecting cancer markers through multiple pathways, providing new ideas for early diagnosis and screening of diseases.

Bimetallic FeOx-TiO2@Carbon hybrid structure materials with notable peroxidase enzyme mimics applied to one-step colorimetric detection of glucose.

FeOx-TiO2@Carbon hybrid structure materials (FeOx-TiO2@CHs) with high peroxidase (POD)-like activity have been prepared by one-pot hydrothermal method. Based on the excellent POD activity of FeOx-TiO2@CHs, one pot colorimetric detection for glucose was constructed by using TMB as substrate with the synergistic reaction of glucose oxidase; the linear range and the limit of detection (LOD) are 25 ~ 1000 and 1.77 µM, respectively. Using this method, the glucose in serum real samples was detected with satisfactory results, and the results are consistent with that of the glucometer method in the hospital. The recovery in diabetic and artificial urine samples was 95.71 ~ 104.67% and 99.01 ~ 103.16%, respectively. The mechanism of the catalytic colorimetric reaction was also investigated by multiple measurements, and the results indicated that superoxide anions (O2•-) between FeOx-TiO2@CHs and substrate play a main role, but a small quantity of hydroxyl radical •OH and singlet oxygen 1O2 is also generated simultaneously. The one-pot reaction method is simple and fast; the detection process only requires a simple mixing, which is suitable for application in special environment.

Highly Active and Robust Catalyst: Co2B-Fe2B Heterostructural Nanosheets with Abundant Defects for Hydrogen Production.

A high-performance and reusable nonnoble metal catalyst for catalyzing sodium borohydride (NaBH4) hydrolysis to generate H2 is heralded as a nuclear material for the fast-growing hydrogen economy. Boron vacancy serves as a flexible defect site that can effectively regulate the catalytic hydrolysis performance. Herein, we construct a uniformly dispersed and boron vacancy-rich nonnoble metal Co2B-Fe2B catalyst via the hard template method. The optimized Co2B-Fe2B exhibits superior performance toward NaBH4 hydrolysis, with a high hydrogen generation rate (5315.8 mL min-1 gcatalyst-1), relatively low activation energy (35.4 kJ mol-1), and remarkable cycling stability, outperforming the majority of reported catalysts. Studies have shown that electron transfer from Fe2B to Co2B, as well as abundant boron defects, can effectively modulate the charge carrier concentration of Co2B-Fe2B catalysts. Density functional theory calculations confirm that the outer electron cloud density of Co2B is higher than that of Fe2B, among which Co2B with high electron cloud density can selectively adsorb BH4- ions, while the electron-deficient Fe2B is favorable for capturing H2O molecules, therefore synergistically promoting the catalytic NaBH4 hydrolysis to produce H2.

Simple enzyme-free detection of uric acid by an in situ fluorescence and colorimetric method based on Co-PBA with high oxidase activity.

In this work, we prepared a simple and low-cost cobalt-doped Prussian blue analog (Co-PBA), which can directly oxidize 10-acetyl-3,7-dihydroxyphenoxazine and 3,3',5,5'-tetramethylbenzidine (TMB) to produce resorufin (ox-AR) with high fluorescent quantum yield and ox-TMB with blue color, respectively, without the need for unstable H2O2. Using the Michaelis-Menten curve and Lineweaver-Burk equation, the Michaelis-Menten constant of Co-PBA and the substrate TMB was found to be 0.033 mM, which was much lower than horseradish peroxidase and other reported nanozymes, showing satisfactory substrate affinity. Uric acid (UA) can cause erosion of the Co-PBA structure, and it significantly reduces the catalytic activity of Co-PBA, resulting in the decrease of the fluorescence emission signal of ox-AR and the absorption signal of ox-TMB. Based on this, a simple, sensitive, and fast fluorescence/colorimetric dual-mode uric acid detection platform was established. The detection range for UA by fluorescence method is 0.625-40 µM, and the detection limit (LOD, S/N = 3) is as low as 0.389 µM. The detection system was applied to serum samples with good recovery and can be used for field detection of UA in biological samples under different environments to meet different needs.

A novel nanoparticle surface-constrained CRISPR-Cas12a 3D DNA walker-like nanomachines for sensitive and stable miRNAs detection.

The CRISPR-Cas system has broad prospects as a new type of nucleic acid signal amplification technology based on the trans-cleavage activity of Cas12a to single-stranded DNA, but the trans-cleavage reaction efficiency is relatively low in solution. In order to overcome this negative factor, a new 3D DNA nanomachine whose CRISPR-Cas12a is limited to the surface of nanoparticles is used for sensitive and stable detection of miRNA. By loading Cas12a activator onto spherical nucleic acid (SNA), the CRISPR-Cas12a activator system on the surface of Au nanoparticles (AuNPs) acts as a walker to carry out continuous recognition-walking-cutting reaction on the surface of AuNPs, which enhances the trans-cleavage activity of Cas12a to SNAs. Benefiting from the confinement effect of spherical nucleic acids surface, a 3D DNA nanomachine has been developed for the detection of miRNA-21, which has achieved high sensitivity and accuracy, and the detection limit is able to reach 8.0 pM. This new 3D DNA walker-like nanomachine provided another insight for future bioanalysis and early clinical diagnoses of disease and liquid biopsy.

A novel label-free universal biosensing platform based on CRISPR/Cas12a for biomarker detection.

The development of a biosensing platform with high sensitivity, high specificity, and low cost for the detection of biomarkers, especially one that is programmable and universal, is critical for disease surveillance and diagnosis, yet it remains a difficulty. Herein, we combined the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system with a fluorescent label-free biosensor platform for sensitive and specific detection of disease-related protein, small molecule and nucleic acid. In this strategy, we designed an exonuclease III-mediated target cycle and released a universal trigger chain to stimulate the enzyme activity of CRISPR/Cas12a for additional signal amplification. The hydrolysis of ssDNA-templated silver nanoclusters (ssDNA-Ag NCs) as the reporter probe resulted in a significant decrease of fluorescence intensity. This biosensing platform can be flexibly used to the sensitive and specific determination of protein, small molecule, or microRNA in biological samples by simply transforming the target recognized sequences in the DNA hairpin. In this work, a new label-free sensing system used the fluorescent ssDNA-Ag NCs as the signal output does not need to be marked in advance and has no background signal. In addition, the method has the advantages of low cost, simple operation and high speed, and provides an innovative idea for the development of a powerful clinical diagnosis tool.

Intracellular Multicomponent Synchronous DNA-Walking Strategy for the Simultaneous Quantification of Tumor-Associated Proteins in a Single Cell.

Single-cell protein analysis is very important for understanding cellular heterogeneity and single-cell biology. However, owing to the extremely low levels of some tumor-associated proteins in individual cells, the absolute quantification of such tumor-associated proteins in a single cell remains a challenge. Herein, an intracellular multicomponent synchronous DNA-walking strategy is proposed for the simultaneous quantification of multiple tumor-associated proteins in a single cell. In this strategy, a nanoprobe based on a DNA walker was designed for the simultaneous signal amplification of nucleolin (NCL) and thymidine kinase 1 (TK1) in a single cell. NCL and TK1 in single cells were simultaneously detected on a microchip platform with detection limits of 1.0 and 0.8 pM, respectively. The results obtained from 20 liver cancer cells (HepG2) and 20 normal hepatocytes (HL-7702) indicated that NCL and TK1 were overexpressed in liver cancer cells. However, the levels of NCL and TK1 in normal hepatocytes are only about one-tenth to one-sixth of those in hepatic carcinoma. Using different nucleic acid aptamers, the proposed strategy can be applied for the analysis of other single-cell proteins and in the early diagnosis of cancer.

A sensitive and facile microRNA detection based on CRISPR-Cas12a coupled with strand displacement amplification.

MicroRNAs (miRNAs) are important biomarkers that are closely associated with certain diseases. The detection of miRNA is critical because it provides the necessary information for Disease Diagnosis. In this study, we achieved miRNA determination by coupling the CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated) system with strand displacement amplification (SDA). In the experiment, miRNA was used as the initiator of SDA, and the activator of Cas12a nuclease activity was amplified by SDA. Subsequently, the unique nuclease activity of Cas12a was exploited to carry out trans cleaving on the ssDNA reporting probe modified with carboxyfluorescein(FAM) and BHQ1(dark Quencher: 480-580 nm) to achieve a signal output. In addition to chain design and reaction simplification, this method is lofty sensitive and selective for the determination of miRNA with a good linear range of 250 fmol·L-1 âˆ¼ 40 pmol·L-1, the detection limit of 150 fmol·L-1 (S/N = 3), and the method showed good recovery in spiked human serum. Overall, this method is expected to be applied to diagnosis with miRNA biomarkers because of its rapidity, high sensitivity, and high selectivity.

A DNAzyme-driven random biped DNA walking nanomachine for sensitive detection of uracil-DNA glycosylase activity.

Highly specific and ultrasensitive detection of uracil-DNA glycosylase (UDG) activity is of great significance for maintaining genomic integrity and medical research of related diseases. Here, we constructed a random DNA walking nanomachine based on a DNAzyme for UDG activity detection on the AuNP (Au nanoparticle) surface. When UDG is present, the U bases in the Y structure are removed, resulting in AP sites, which will be cleaved by Endo-IV to generate a 3' concave end for Exo-III, causing the locking strand of the DNAzyme to be completely hydrolyzed by the Exo-III and release the walking strand to randomly pair with the substrate strand on the AuNP surface; then, the walking strand exerts its cleavage activity with the assistance of Mg2+ to cleave the substrate strand and keep the fluorophore 6-carboxyfluorescein (FAM) away from the surface of the AuNP, which restores the fluorescence signal of this system. In this way, sensitive detection of UDG can be realized, and the detection limit is as low as 3.69 × 10-6 U mL-1. In addition, we found that this method is highly specific to UDG and can be used to detect UDG specifically in complex samples, which has certain application prospects in biomedical research and clinical diagnosis related to UDG.

A mitochondria-targeted ratiometric fluorescent nanoprobe for imaging of peroxynitrite in living cells.

Peroxynitrite (ONOO-) is a series of basic biological oxidants involved in physiological and pathological processes. The detection of ONOO- in biological systems has been challenging due to its extremely short half-life and low steady-state concentration. In this work, a ratiometric fluorescent nanoprobe for ONOO- was constructed by coupling covalently of graphene quantum dots (GQDs) with cyanine 5.5 (Cy5.5). This nanoprobe (GQD-Cy5.5) could selectively accumulate in mitochondrial, appears two strong fluorescence emission peaks at 520 and 694 nm. In the presence of ONOO-, the intensity of fluorescence emission peak at 520 nm increased and the intensity of fluorescence emission peak at 694 nm decreased. The ratio (F520 nm/F694 nm) of fluorescence intensity at two emission peaks had a good linear relationship with the concentration of ONOO- in the range of 0-6.0 µM, and the detection limit was 0.03 µM. The excellent properties of the nanoprobe enable its applications in the ratiometric fluorescence imaging of endogenous ONOO- in cell mitochondria.

Sensitive detection of microRNA using a label-free copper nanoparticle system with polymerase-based signal amplification.

The abnormal expression of microRNAs (miRNAs) has been reported in many diseases, so it is of great interest to develop simple and accurate methods for the detection and analysis of miRNA expression. We have developed a novel biosensor to detect miRNAs. This method is based on a polymeric double-stranded DNA (dsDNA) copper nanoparticle (CuNP) template that is synthesised by a polymerase. When Cu2+ and ascorbic acid are added to the system, the dsDNA template (which is rich in A-T bases) promotes the formation of CuNPs, resulting in high fluorescence intensity. This system provides sensitive analysis of miRNA expression with a limit of detection down to 17.8 pmol/L, due to significant changes in the fluorescence signal of the system before and after the addition of the target. The linear range between F0-F and concentration of miR-122 is 80.0 pmol/L to 4.50 nmol/L, and the recovery rate in spiked HepG2 cell lysates is 93.33-102.53%. This method expands the applications of fluorescent DNA-CuNPs in the field of biosensor analysis, and can be used to detect and analyse any miRNA marker by changing the target recognition sequence. Graphical abstract A label-free dsDNA-CuNP-based and enzyme-assisted signal amplification method for microRNA is constructed.

Ratiometric fluorescent 3D DNA walker and catalyzed hairpin assembly for determination of microRNA.

Using 6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detection was constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. The dual emission ratio fluorescent signal with a single excitation wavelength was used as the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experiment is reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488 nm and the maximum fluorescence emission wavelength of 520 nm and 580 nm, respectively. It has a good linear response at a microRNA concentration range of 156.0 pM ~ 7.00 nM and a detection limit of 42.94 pM. This strategy has been successfully applied to detect microRNAs in spiked serum samples. Graphical abstract Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis.

Dissolution reconstruction of electron-transfer enhanced hierarchical NiSx-MoO2 nanosponges as a promising industrialized hydrogen evolution catalyst beyond Pt/C.

An industrial electro-catalyst obliges three essential features, such as scalability, generating high current density at low overpotential, and long-term stability. Herein, we tackle those challenges using NiSx-MoO2 nanosponges on carbon cloth based hydrogen evolution catalyst. The target catalyst was synthesized through a series of simple and scalable methods, including dissolution, reconstruction, and chemical vapor deposition. The optimized NiSx-MoO2/CC catalyst exhibits a superior hydrogen evolution catalytic activity far better than commercial Pt/C meanwhile surpasses widely used industrial Raney Ni catalyst by many aspects, namely lower overpotential at 500 mA cm-2 current density and smaller Tafel plot in 30 wt% KOH solution. This excellent electrocatalytic activity is attributed to enhanced mass transfer and faster reaction kinetics due to the unique hierarchical porous structures, as well as the synergistic electron transfer effect between the two components of NiSx and MoO2 species. This work may provide a new strategy for the design of better hydrogen evolution catalyst for industrial application.

Template synthesis of two-dimensional ternary nickel-cobalt-nitrogen co-doped porous carbon film: Promoting the conductivity and more active sites for oxygen reduction.

Rational design of a stable, highly active non-precious metal-based electrocatalysts for oxygen reduction reaction (ORR) is vitally important for industrial application of fuel cells technology. As a potential alternative of Pt/C catalyst, two-dimensional (2D) porous carbon materials are widely investigated due to the highly accessible surface area and active sites, wherein carbon films doped with a plurality of metals and non-metal elements are rarely reported due to an uncontrollable synthesis process. Here, a bi-metallic (NiCo alloy nanoparticles) and nonmetallic (N) co-doped porous carbon film (Ni-Co-N@CF) is fabricated by a simple controllable and scalable strategy comprising the synthesis of NiCo alloy nanoparticles, modification of organic molecules, and high-temperature carbonization process. The optimized Ni-Co-N@CF catalyst shows an excellent ORR electrocatalytic activity with a larger electrochemically active surface area (2.31 m2 g-1), a higher half-wave potential (0.86 V) and a lower diffusion limited current density (-4.43 mA cm-2) than all the prepared control catalysts. Moreover, the designated catalyst also exhibits high durability and superior methanol tolerance in alkaline media, significantly better than the commercial Pt/C (20 wt%). The superior ORR performance is attributed to the synergetic interactions of ternary doping of Ni/Co/N in the 2D film skeleton, which not only greatly enhances conductivity but also provides more Co-N active sites.

Detection of microRNA using enzyme-assisted amplifying and DNA-templated silver nanoclusters signal-off fluorescence bioassay.

A Simple and fast analysis strategy of fluorescence quenching based on DNA-templated silver nanoclusters was developed for detection of miR-122 related to diseases such as human liver. We used Exo III to cleave the silver cluster template and assist in the DNA-RNA complex cycle. When the target is absent, the silver cluster template remains intact, and DNA-AgNCs are generated under the action of AgNO3/NaBH4, producing a strong background fluorescence signal. Once the target is added, the site of the Exo III occurs after a series of hybridization cycles, the Exo III acts, the template DNA is continuously hydrolyzed, and the fluorescence intensity of the system is significantly reduced. By comparing the changes in the fluorescence signal, we found that this strategy has good sensitivity and the detection limit is as low as 84.0 pM. The strategy also has excellent discriminating ability and good selectivity, it can provide a persuasive reference for the early diagnosis of liver cancer and hepatitis.

Ultrasensitive detection of microRNA-21 based on electrophoresis assisted cascade chemiluminescence signal amplification for the identification of cancer cells.

Rapid and accurate detection of microRNA content in cells is of great significance. Here, an ultrasensitive microchip electrophoresis (MCE) method based on cascade chemiluminescence (CL) signal amplification was developed for the detection of microRNA-21 in cells. In this method, horseradish peroxidase labeled DNA was used as a signal probe, which could induce CL signal by the reaction of luminol and H2O2. Combining with two cyclic enzyme digestion reactions by T7 exonuclease, a large number of signal probes were degraded. By using MCE-CL as a separation and detection platform, an amplified CL signal peak was achieved. The developed MCE-CL method can detect miR-21 at a concentration as low as 1.0 × 10-15 M, which was enhanced by six orders of magnitude compared with those of conventional MCE-CL assay. This method has been applied for the detection of microRNA-21 in cell lysate, which show that there were significant differences of miR-21 among different types of cells, and the content in cancer cells was much higher than that in normal cells, which can be used for the identification of cancer cells. Therefore, the proposed method held great application potential in early diagnosis of tumor and biomedical research.

Fluorometric determination of microRNA-122 by using ExoIII-aided recycling amplification and polythymine induced formation of copper nanoparticles.

The authors describe a method for the determination of microRNA-122 by using terminal deoxynucleotidyl transferase (TdT). It is based on the use of polythymine and exonuclease III-aided cycling amplification. A 3'-phosphorylated hairpin probe 1 (H1) and a hairpin probe 2 (H2) were designed. In the presence of the microRNA, hybridization and enzymatic cleavage will occur and produce lots of 3'-hydroxylated ssDNA which can be tailed by TdT and converted into long polythymine (polyT) sequences. These can be used to synthesize copper nanoparticles (CuNPs) with fluorescence excitation/emission maxima at 350 nm/630 nm. This method shows good selectivity and high sensitivity with a linear response in the 1.00 × 102 fM and 1.00 × 106 fM microRNA concentration range and a 44 fM limit of detection. It was successfully applied to determination of microRNA in spiked serum samples. Graphical abstract A label-free and highly sensitive fluorometric method is described for the assay of microRNA on the basis of target-triggered two-cycle amplification and combining with terminal TdT. It produces a series superlong polyT that can be used for synthesis of copper nanoclusters (CuNCs) displaying red fluorecence.

Fluorescence polarization gene assay for HIV-DNA based on the use of dendrite-modified gold nanoparticles acting as signal amplifiers.

The authors describe a fluorescence polarization assay for HIV-DNA. It is based on the use of gold nanoparticles (AuNPs) modified with DNA dendritic macromolecules that act as signal amplifiers. In the presence of HIV-DNA, the AuNP-DNA dendritic macromolecules and fluorescently labeled DNA probe combine with HIV-DNA in a sandwich format to form a conjugate. This reaction slows down the rotational speed of the labeled DNA probe because of the increase of molecular weight and volume. This increases fluorescence polarization and the sensitivity of the system. The relative fluorescence polarization values increase linearly in the 150 pM to 6 nM HIV-DNA concentration range, with a 73 pM detection limit. The results show this amplification strategy to be most useful for ultrasensitive determination of oligonucleotides by means of fluorescence polarization. Graphical abstract Schematic of a novel fluorescence polarization assay for the HIV-DNA. Ultrasensitive detection is accomplished by using AuNP-DNA dendritic macromolecules as signal amplification factor.

Rapid and label-free fluorescence bioassay for microRNA based on exonuclease III-assisted cycle amplification.

The quantitative analysis of microRNA is extremely important in biological research and clinical diagnosis due to the relationship between microRNA and disease. In this study, we reported a new assay for the rapid and simple detection of microRNA based on G-quadruplex and exonuclease III (ExoIII) dual signal amplification. We specifically designed two hairpins with G-quadruplex sequence. In the absence of a target, the G-quadruplex sequences are enclosed in the hairpin and fluorescence signal shut down. However, when a target is added, the dual cycle is carried out because two hairpins are digested and X and Y sequences are released under the action of ExoIII. Then, these released sequences form the G-quadruplex sequence, and N-methylmorpholine (NMM) is embedded in the G-quadruplex to produce strong fluorescence. The linear range is from 2.5 × 10-10 to 4 × 10-9 mol L-1 with a low detection limit of 6 pM. Compared to some of the previous strategies, this bioassay needs only a simple one-step reaction, and is easy for realizing the rapid detection of microRNAs. The time required for the entire analysis is only 1 hour. In addition, this bioassay has good specificity and can be applied to the actual samples.