Preparation and application of high-brightness red carbon quantum dots for pH and oxidized L-glutathione dual response.

Carbon dots (CDs) with red fluorescence emission are highly desirable for use in bioimaging and trace- substance detection, with potential applications in biotherapy, photothermal therapy, and tumor visualization. Most CDs emit green or blue fluorescence, thus limiting their applicability. We report a novel fluorescent detection platform based on high-brightness red fluorescence emission carbon dots (R-CDs) co-doped with nitrogen and bromine, which exhibit pH and oxidized L-glutathione (GSSG) dual-responsive characteristics. The absolute quantum yield of the R-CDs was as high as 11.93%. We discovered that the R-CDs were able to detect acidic pH in live cells and zebrafish owing to protonation and deprotonation. In addition, GSSG was detected in vitro over a broad linear range (8-200 µM) using the R-CDs with excitation-independent emission. Furthermore, cell imaging and bioimaging experiments demonstrated that the R-CDs were highly cytocompatible and could be used as fluorescent probes to target lysosomes and nucleolus. These studies highlight the promising prospects of R-CDs as biosensing tools for bioimaging and trace-substance detection applications.

Nitrogen-doped orange emitting carbon dots for ß-carotene detection and lysosomal imaging.

ß-Carotene is a natural antioxidant that has an indispensable effect on the growth and immunity of the human body. For intracellular and in vitro detection of ß-carotene, N-doped carbon quantum dots (O-CDs) were prepared by co-heating carbonization of 1,5-naphthalenediamine and nitric acid in ethanol solvent for 2 h at 200 °C. O-CDs have longer wavelength orange light emission, with an optimal excitation peak of 470 nm and an optimal emission peak of 590 nm. According to the principle of the internal filtering effect on which the detection system is based, O-CDs present a good linear relationship with ß-carotene within a wide range of 0-2000 µM, and the R2 coefficient of the linear regression equation is 0.999. In addition, O-CDs showed targeting of lysosomes in cell imaging and could be used to detect intracellular lysosomal movement. These experiments show that O-CDs can be used for in vivo and in vitro detection of ß-carotene and can serve as a potential substitute to commercial lysosome targeting probes.

Injectable Carbon Dots-Based Hydrogel for Combined Photothermal Therapy and Photodynamic Therapy of Cancer.

Hydrogel has been widely used in modern biotherapeutics due to its excellent biocompatibility, degradability, and high drug loading capacity. Among them, the construction of a phototherapy system including photosensitizer and hydrogel has aroused great interest in tumor therapy. Unfortunately, complex modifications are necessary to integrate different photosensitizers into the hydrogel. In this work, an injectable hydrogel was proposed by the Schiff base reaction between HA-CHO and carbon dots (CDs), which can realize PTT and PTT simultaneously. Notably, the CDs with rich -NH2 can be used not only as a photosensitizer but also as an efficient cross-linking agent for the Schiff base reaction to form a hydrogel network. The CD@Hydrogel with outstanding biosafety showed a high antitumor effect after 660 nm laser irradiation in in vitro and in vivo experiments. In summary, the CD@Hydrogel can not only realize PTT and PDT synergistic treatment under one light source but also act as a cross-linking agent to react with HA-CHO to form hydrogel, which is simple and efficient, providing a new strategy for cancer phototherapy.

Establishment of a Monoclonal Antibody-Based Enzyme-Linked Immunosorbent Assay to Measure Soluble B7-H5 in Patients with Cancer.

B7-H5, an immune checkpoint molecule, is markedly upregulated in multiple cancers and plays an important role in tumor progression and immune escape. However, the expression and significance of soluble B7-H5 (sB7-H5) in cancer remain unclear. Herein, we generated two novel mouse anti-human B7-H5 monoclonal antibodies (mAbs) 2E5 and 7B10, which had different epitopes. Based on the two mAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) system was developed. Using this ELISA, we found that compared with healthy controls (HCs), sB7-H5 levels were significantly increased in the serum of patients with gastric cancer (GC), colorectal cancer (CRC), and lung cancer (LC) and were associated with TNM stage and metastasis. Receiver operating characteristic (ROC) curve analysis showed that sB7-H5 has diagnostic value for GC, CRC, and LC. Collectively, our findings delineate that sB7-H5 may be used as a predictor for diagnosis of cancer and a potential therapeutic target for cancer treatment.

Roll-to-Roll DNA Nanomachine for Ultrasensitive Electrochemical Determination of miRNA.

MicroRNAs (miRNAs) are a family of endogenous noncoding RNAs with the functions of gene regulation, which serve as promising markers for a range of diseases such as diabetic foot ulcers, cancers, etc. In this work, we engineered a roll-to-roll DNA nanomachine for highly sensitive electrochemical detection of miRNA. A dumbbell-structured DNA probe could be transitioned to be wheel-structured conformation upon target recognition, which rolls around track strands on the surface of gold nanoparticles (AuNPs) in the presence of nicking endonuclease. The resulting single strands on AuNPs are activated for the second round of rolling at the DNA-modified electrode interface, leading to the variation of electrochemical responses. The roll-to-roll amplification behavior allows a wide detection range with a limit of detection as low as 10 aM. The practicability is also demonstrated by the application in human serum samples with satisfactory results. It is expected that the proposed electrochemical method offers a new paradigm to develop miRNA assays based on DNA nanotechnology.

Non-doped and non-modified carbon dots with high quantum yield for the chemosensing of uric acid and living cell imaging.

Carbon dots without heteroatoms-doping and surface modifications were designed to be a novel chemosensing strategy on the quantitative detection of uric acid (UA) with the aid of uricase-induced enzymatic reaction and Fenton reaction. In this work, ascorbic acid (AA)-derived carbon dots (A-CDs) were prepared in the mixture of ethanol and water via one-step hydrothermal synthesis at a relatively low temperature (120 °C) for 10 h. The resultant A-CDs were proved to be excitation-independent. When excited at the wavelength of 420 nm, the nanodots displayed green fluorescence (535 nm) which was then linearly quenched as UA concentration increased in the range of 0-56 µM, according to which the detection limit was calculated to be 0.49 µM. With regards to the excellent sensitivity and selectivity to UA, real sample assay was performed on the A-CDs detection system, which provided relatively reliable recoveries of UA contained in human serum/urine. Besides, in view of the high quantum yield, the A-CDs were applied to live-cell imaging assay and were considered to become an alternative tracer tool in biomedical imaging.

Starch-Based Carbon Dots for Nitrite and Sulfite Detection.

Nitrite and sulfite play important roles in human health and environmental science, so it is desired to develop a facile and efficient method to evaluate NO2 - and SO3 2- concentrations. In this article, the use of green alternatives with the potential of multi-functionality has been synthesized to detect nitrite and sulfite based on fluorescent probe. The carbon dots (CDs) with starch as only raw materials show fluorescence turn "on-off-on" response towards NO2 - and SO3 2- with the limits of detection of 0.425 and 0.243 µÐœ, respectively. Once nitrite was present in the solution, the fluorescence of CDs was quenched rapidly due to the charge transfer. When sulfite was introduced, the quenching fluorescence of CDs was effectively recovered because of the redox reaction between NO2 - and SO3 2-, and thus providing a new way for NO2 - and SO3 2- detection. Owing to their excellent analytical characteristics and low cytotoxicity, the "on-off-on" sensor was successfully employed for intracellular bioimaging of NO2 - and SO3 2-.

Red-emissive carbon nanodots for highly sensitive ferric(III) ion sensing and intracellular imaging.

Ferric(III) ions (Fe3+) are one of the most abundant metal ions in environmental and biological systems. The determination of Fe3+ has attracted great attention for healthcare concerns. In this work, we have developed a novel fluorescence method for the sensing and intracellular imaging of Fe3+ based on the prepared red-emissive carbon nanodots. The nanoprobes are synthesized via a microwave method using ammonium fluoride and o-phenylenediamine as carbon precursors, which exhibit excellent optical properties and low toxicity. More importantly, the carbon nanodots show high selectivity towards Fe3+ against other interfering ions. The sensitivity is also high with the limit of detection as low as 0.05 µM. Meanwhile, the carbon nanodots have been successfully used for fluorescence imaging of cells and could be quenched by intracellular Fe3+. These results suggest that the red-emissive carbon nanodots have diverse potential utilities in biomedical fields.

Riboflavin-based carbon dots with high singlet oxygen generation for photodynamic therapy.

Photodynamic therapy, as an effective treatment for superficial tumors, has attracted more and more attention. The development of safe, biocompatible and in vivo photosensitive materials is helpful to promote photodynamic therapy. Here we report green fluorescent carbon quantum dots prepared from a natural vitamin, riboflavin (VB2), as a photosensitizer. The VB2-based carbon dots have excellent water solubility and biocompatibility, and their singlet oxygen generation ability is much stronger than that of riboflavin itself. Through endocytosis, the carbon dots can easily enter the cells and show bright green fluorescence. In vivo experiments show that after photodynamic therapy the carbon dots can significantly inhibit the growth of tumors, and will not have toxic and side effects on other organs.

Superior reducing carbon dots from proanthocyanidin for free-radical scavenging and for cell imaging.

The presence of excessive ROS can cause much harm to the human body and can even cause diseases. Therefore, it is important to detect and remove ROS, but there is no ideal method available for this at present. In this research, using procyanidins, a type of plant extract with strong reducibility, as raw materials, fluorescent carbon dots (CDs) were prepared by a hydrothermal method. The proanthocyanidin-based carbon dots (PCDs) emit a light-green colored light under UV irradiation. The PCDs retain the strong reducibility of procyanidins and are highly water-soluble compared with procyanidins. The PCDs, in addition to having good biocompatibility, also have the superior properties of radical scavenging activity and cell imaging. In in vitro experiments, 1,1-diphenyl-2-picrylhydrazyl (DPPH; 100 µM) was reduced by 30% when PCDs were added up to a concentration of 87.5 µg mL-1. At the same time, the fluorescence quenching correlates with the concentration of hypochlorite and hydrogen peroxide and has a good linearity in the range of 250-2250 nM and 60-180 µM with a detection limit of 3.676 nM and 0.602 µM, respectively. Based on the previously described advantages, PCDs have potential as a biomedicine.

Green Synthesis of Lutein-Based Carbon Dots Applied for Free-Radical Scavenging within Cells.

Reactive oxygen species (ROS) in the body play an important role in various processes. It is well known that harmful high levels of ROS can cause many problems in living organisms in a variety of ways. One effective way to remove intracellular ROS is to use reducing materials that can enter the cell. Herein, we developed a strong reducing carbon nano-dot from a natural product, lutein, as an initial raw material. This is a hydrothermal synthesis method with the advantages of simplicity, high yield, mild reaction conditions, and environmental friendliness. The prepared carbon dots exhibit bright blue fluorescence, and have good water solubility and biocompatibility. In particular, the carbon dots can easily enter the cell and effectively remove ROS. Therefore, the carbon dots are thought to protect cells from oxidative damage by high levels of ROS.

Biomimetic immunomagnetic gold hybrid nanoparticles coupled with inductively coupled plasma mass spectrometry for the detection of circulating tumor cells.

Immunomagnetic beads are important tools for the isolation and detection of circulating tumor cells (CTCs). However, the current immunomagnetic bead technique provides poor CTC separation purity due to nonspecific binding of background cells. Furthermore, immunomagnetic beads have not been appropriately functionalized for enabling CTC analysis and quantification. In this work, bimetallic magnetic gold nanoparticles were prepared and coated with leukocyte membranes to form leukocyte membrane-camouflaged nanoparticles. After conjugation with the antibody of epithelial cell adhesion molecule (EpCAM), the biomimetic immunomagnetic gold nanoparticles (CM-Fe3O4@Au-Ab) showed a high specific recognition ability on mock (EpCAM-positive) CTCs and a reduced interaction with leukocytes. We subsequently optimized the conditions for CTC separation, including the concentration of nanoparticles and the incubation time. Under the optimized conditions, CM-Fe3O4@Au-Ab exhibited high CTC capture efficiency with negligible background cell binding in mock clinical blood samples. More importantly, gold probes were tagged on the surface of these separated CTCs. When coupled with ICP-MS analysis, the number of CTCs and gold signals exhibited a good linear relationship, and a low limit of detection was obtained, enabling us to estimate the number of CTCs in blood samples. Hence, we expected that CM-Fe3O4@Au-Ab could provide an opportunity to surmount the limitations of current CTC detection.

Author Correction: The Transmembrane Serine Protease HAT-like 4 Is Important for Epidermal Barrier Function to Prevent Body Fluid Loss.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Duplex-specific nuclease assisted miRNA assay based on gold and silver nanoparticles co-decorated on electrode interface.

miRNAs are small non-coding RNAs for gene regulation, which serve as promising biomarkers for the diagnosis of certain diseases. In this contribution, we have proposed a convenient electrochemical biosensing strategy based on the interaction between DNA modified gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs). In principle, citrate capped AuNPs and AgNPs can be co-decorated on the electrode successively. However, with the modification of DNA on AuNPs surface, a strong negative layer is formed. AuNPs@DNA modified electrode could then inhibit subsequent adsorption of AgNPs due to the electrostatic repulsion and steric hindrance effect. As a result, electrochemical response from AgNPs is significantly decreased. On the other hand, in the presence of target miRNA, DNA on AuNPs hybridizes with miRNA and can thus be cyclically digested by duplex-specific nuclease (DSN). Without the shield of DNA, AgNPs can be relaunched at the AuNPs modified electrode. By analyzing the silver stripping peak, highly sensitive detection of miRNA can be achieved. This biosensor exhibits the limit of detection as low as 0.62 fM and a broad linear range from 1 fM to 1 pM. It may hold great potential utility for miRNA assay in the applications of biomedical researches and early clinical diagnosis.

Triple-Input Molecular AND Logic Gates for Sensitive Detection of Multiple miRNAs.

Abnormal miRNA expressions are closely related to the occurrence and development of cancers. It is of great significance to monitor miRNA expression levels for early diagnosis and therapy of the diseases. This study presents two independent colorimetric strategies for simultaneously monitoring multiple miRNAs based on cross-linking or non-cross-linking aggregations of gold nanoparticles (AuNPs). By introducing a Y shaped DNA structure and two types of DNA modified AuNPs, a triple-input DNA AND logic gate is facilely developed with the cross-linking aggregation of AuNPs as the signal output. To improve the sensitivity and shorten reaction time, the logic gate is modified by further employing a three DNA strands formed duplex and hybridization chain reaction. Non-cross-linking aggregation of AuNPs is used to evaluate the concentration of initial miRNA inputs. This strategy does not require DNA modification of AuNPs and ultrahigh sensitivity is achieved with the amplification of hybridization chain reaction. The present work may provide powerful tools for multiple miRNAs diagnostics and inspire further development of DNA based logic gates.

Ectopic expression of human airway trypsin-like protease 4 in acute myeloid leukemia promotes cancer cell invasion and tumor growth.

Transmembrane serine proteases have been implicated in the development and progression of solid and hematological cancers. Human airway trypsin-like protease 4 (HAT-L4) is a transmembrane serine protease expressed in epithelial cells and exocrine glands. In the skin, HAT-L4 is important for normal epidermal barrier function. Here, we report an unexpected finding of ectopic HAT-L4 expression in neutrophils and monocytes from acute myeloid leukemia (AML) patients. Such expression was not detected in bone marrow cells from normal individuals or patients with chronic myeloid leukemia, acute lymphocytic leukemia and chronic lymphocytic leukemia. In AML patients who underwent chemotherapy, persistent HAT-L4 expression in bone marrow cells was associated with minimal residual disease and poor prognostic outcomes. In culture, silencing HAT-L4 expression in AML-derived THP-1 cells by short hairpin RNAs inhibited matrix metalloproteinase-2 activation and Matrigel invasion. In mouse xenograft models, inhibition of HAT-L4 expression reduced the proliferation and growth of THP-1 cell-derived tumors. Our results indicate that ectopic HAT-L4 expression is a pathological mechanism in AML and that HAT-L4 may be used as a cell surface marker for AML blast detection and targeting.

Increased Neutrophil Activation and Plasma DNA Levels in Patients with Pre-Eclampsia.

Pre-eclampsia (PE) is a chronic inflammatory disease in pregnancy, which is associated with enhanced blood coagulation and high thrombotic risk. To date, the mechanisms underlying such an association are not fully understood. Previous studies reported high levels of plasma deoxyribonucleic acid (DNA) in PE women, but the cellular source of the circulation DNA remains unknown. In this study, we tested the hypothesis that activated neutrophils undergoing cell death, also called NETosis, may be responsible for the elevated plasma DNA levels in PE women. We analysed plasma samples from non-pregnant, normal pregnant and PE women and found high levels of double-stranded DNA, myeloperoxidase (an abundant neutrophil granular enzyme) and histones (the major nucleosome proteins) in PE-derived samples, indicating increased NETosis in the maternal circulation. The high plasma DNA levels positively correlated with enhanced blood coagulation in PE women. When isolated neutrophils from normal individuals were incubated with PE-derived plasma, an elevated NETosis-stimulating activity was detected. Further experiments showed that endothelial micro-particles, but not soluble proteins, in the plasma were primarily responsible for the NETosis-stimulating activity in PE women. These results indicate that circulating micro-particles from damaged maternal endothelium are a potent stimulator for neutrophil activation and NETosis in PE women. Given the pro-coagulant and pro-thrombotic nature of granular and nuclear contents from neutrophils, enhanced systemic NETosis may represent an important mechanism underlying the hyper-coagulability and increased thrombotic risk in PE.

A novel mode of DNA assembly at electrode and its application to protein quantification.

Sensitive and specific detection of protein is of great significance for early diagnosis and prognosis of many diseases. However, great challenges remain unsolved including relative low sensitivity, high cost, long testing time, complicated instrument and laborious operation. To improve the performance of protein detection methods, development of fine reaction interface for recognition and signal amplification is of great importance. In this work, we construct a novel mode of DNA assembly at electrode interface based on a tripodal surface anchor and an electrochemical aptasensor for protein assay is developed. The orientation of the immobilized DNA is optimized, which promises the efficiency of protein recognition. In addition, hybridization chain reaction is employed for further signal amplification. Therefore, this detection method shows high sensitivity with excellent specificity. The strategy can be universally applicable by simply modifying the sequences of used DNA probes.