A sensitive intelligent point-of-care test method for tert-butylhydroquinone in edible oil via a test strip with a smartphone.

Tert-butylhydroquinone (TBHQ) is easily overused or illegally added to edible oil and attracts a growing concern because of its cytotoxic, liver-damaging, and carcinogenic effects. Thus, a sensitive and intelligent point-of-care testing (iPOCT) method is developed to fulfill the on-site monitoring. This iPOCT method depended on a fluorescent immunochromatographic assay within 15 min. Under optimization, the limit of quantification (LOQ) was calculated as 0.03 µg mL-1. The iPOCT method provided a low limit of detection (LOD) of 0.02 µg mL-1, a wide linear range of 0.03-100 µg mL-1, and great selectivity. Recoveries by the spiking experiments ranged from 97.4% to 103.5% with relative standard deviations (RSDs) of 2.4%-4.9% in soybean, peanut, rapeseed, and corn oil samples. The results showed that the iPOCT method is highly consistent with the high-performance liquid chromatography (HPLC) method.

Simultaneous detection of two subtypes of extracellular vesicles using ultrabright fluorescent nanosphere-based test strips.

In recent years, the cargo profiles of extracellular vesicles (EVs), which were inherited from their parent cells, have emerged as a reliable biomarker for liquid biopsy (LB) in disease diagnosis, prognosis, and treatment monitoring. EVs secreted by different cells exhibit distinct characteristics, particularly in terms of disease diagnosis and prediction. However, currently available techniques for the quantitative analysis of EV cargoes, including enzyme-linked immunosorbent assay (ELISA), cannot specifically identify the cellular origin of EVs, thus seriously affecting the accuracy of EV-based liquid biopsy. In light of this, we here developed ultrabright fluorescent nanosphere (FNs)-based test strips which have the unique capability to specifically assess the levels of PD-L1-positive EVs (PD-L1+ EVs) derived from both tumor cells and immune cells in bodily fluids. The levels of PD-L1+ EV subpopulations in human saliva were quantified using the ultrabright fluorescent nanosphere-based test strips with more convenience and higher efficiency (detection time <30 min). Results demonstrated that the fluorescence intensity of the test line exhibited a good linear relationship respectively with the PD-L1 levels of tumor cell- (R2 = 0.993) and immune cell-derived EVs (R2 = 0.982) in human saliva. By assessing the levels of PD-L1+ EV subpopulations, our test strips hold immense potential for advancing the application of PD-L1+ EV subpopulation-based predictions in tumor diagnosis and prognosis evaluation. In summary, by integrating the benefits of FNs and lateral flow chromatography, we here provide a strategy to accurately measure the cargo levels of EVs originating from diverse cell sources in bodily fluids.

NIR-II fluorescent Ag2Se polystyrene beads in a lateral flow immunoassay to detect biomarkers for breast cancer.

Fluorescent lateral flow immunoassay (LFA), one tool in point of care testing (POCT) systems for breast cancer, has attracted attention because it is quick, simple, and convenient. However, samples and the constituent material exhibit autofluorescence in the visible region, which is a very large obstacle in the development of fluorescent LFAs. The autofluorescence of biological samples is scarcely found in the second near-infrared (NIR-II) range and samples scatter and absorb less NIR-II light than visible light. Here, we report an NIR-II QD-LFA platform using the NIR-II fluorescent Ag2Se quantum dots (QDs) with 1020 nm emission encapsulated into polystyrene beads as fluorescent probes. The NIR-II LFA platform was established to detect breast cancer tumour markers (CEA and CA153) within 15 min with a low limit of detection (CEA: 0.768 ng mL-1, CA153: 1.192 U mL-1), high recoveries (93.7% ~ 108.8%), and relative standard deviations (RSDs) of less than 10%. This study demonstrated the potential of NIR-II Ag2Se polystyrene beads as a fluorescent probe in LFA for rapid and accurate identification of biomarkers. They are suited for use in professional situations.

NIR quantum dot construction of a fluorescence anisotropy signal amplification biosensor for sensitive, rapid and separation-free detection of dopamine in serum.

Dopamine (DA) is an important small-molecule neurotransmitter, which is closely related to the development of many neurological diseases and has received increasing attention in the diagnosis of neurological diseases. Currently, the assays of the detection of dopamine such as electrochemical and colorimetric methods have low sensitivity, poor selectivity and susceptibility to interference, which limit the accurate quantification of dopamine. Fluorescence anisotropy immunoassay is a traditional analytical method in which the quantification is based on the change in fluorescence anisotropy values observed when fluorescence molecules are bound to a certain volume and mass of the material. Since dopamine is a small molecule with small volume and mass, we took advantage of the good photostability of the second near-infrared window (NIR-II) quantum dots (QDs) and the low spontaneous interference of the substrate, and designed a biosensor dopamine fluorescence anisotropy probe streptavidin biosensor (DFAP-SAB) based on the NIR-II QDs combined with streptavidin signal amplification to achieve rapid and separation-free detection of dopamine in human serum. The detection signal has a good linearity between 50 nM and 3000 nM with a detection limit of 11.2 nM. The application of NIR-II QDs provides the possibility of biosensor applications for complex samples. The construction of the streptavidin signal amplification device provides a new idea for small molecule detection.

Recapitulating Developmental Condensation and Constructing Self-organised Cartilaginous Tissue for Cartilage Regeneration.

OBJECTIVE: To develop a novel chondrocyte condensation culture strategy recapitulating developmental condensation and construct self-organised cartilaginous tissue for cartilage regeneration. METHODS: Cell-condensation aggregate (CCA) was generated using the condensation culture method by sequential cell seeding. The chondrification capacities and biocompatibilities of CCA were assessed by comparison with the cell-scaffold complex (CSC), which was constructed by cell-scaffold coculture. Preclinical studies including implantation into nude mice subcutaneously and cartilage defect repair in rabbits were performed. RESULTS: CCA constructed by condensation culture exhibited a morphology of self-organised cartilaginous tissue. Meanwhile, the condensation culture inhibited or abolished expression of HOX genes including HOXC4 and HOXD8, which was partially consistent with developmental HOX gene expression patterns and associated with enhanced regeneration capacities. Compared with CSC, CCA showed a higher capacity for chondrification and regeneration of rabbit cartilage defects. CONCLUSION: The therapeutic assessments indicate that CCA is an efficient therapeutic tool for cartilage regeneration, providing a new strategy for tissue engineering by mimicking developmental events.

A near-infrared-II fluorescence anisotropy strategy for separation-free detection of adenosine triphosphate in complex media.

Fluorescence anisotropy (FA) has been widely applied for detecting and monitoring special targets in life sciences. However, matrix autofluorescence restricted its further application in complex biological samples. Herein, we report a near-infrared-II (NIR-II) FA strategy for detecting adenosine triphosphate (ATP) in human serum samples and breast cancer cell lysate, which employed NIR-II fluorescent Ag2Se quantum dots (QDs) as tags to reduce matrix autofluorescence effect and applied graphene oxide (GO) to enhance fluorescence anisotropy signals. In the presence of ATP, the recognition between NIR-II Ag2Se QDs labeled aptamer (QD-pDNA) and ATP led to the release of QD-pDNA from GO, resulting in the obvious decrease of FA values. ATP could be quantitatively detected in concentrations ranged from 3 nM to 2500 nM, with a detection limit down to 1.01 nM. This study showed that the developed NIR-II FA strategy could be applied for detecting targets in complex biological samples and had great potential for monitoring interactions between biomolecules in biomedical research.

Target-modulated sensitization of upconversion luminescence by NIR-emissive quantum dots: a new strategy to construct upconversion biosensors.

We herein used Ag2Se quantum dots (QDs) as a target-modulated sensitizer for upconversion nanoparticles (UCNPs) and the target thrombin as the sensitizing switch to construct a biosensor, circumventing the limited luminescence resonance energy transfer (LRET) efficiency of UCNPs, with enhanced signal-to-background ratio (SBR) and assay sensitivity.

Gd-DTPA-coupled Ag2Se quantum dots for dual-modality magnetic resonance imaging and fluorescence imaging in the second near-infrared window.

Near-infrared (NIR) fluorescent quantum dots (QDs) are ideal platforms to fabricate multifunctional contrast agents for multimodal imaging. Herein, second near-infrared window fluorescent (NIR-II) Ag2Se QDs were coupled with gadopentetate dimeglumine injection (Gd-DTPA) for dual-modality T1-weighted magnetic resonance (MR) imaging and fluorescence imaging. In vitro experiments suggested that the prepared Ag2Se-Gd QDs exhibit low cytotoxicity, remarkable T1-weighted MR imaging, and fluorescence imaging contrast properties. In vivo experiment results showed that Ag2Se-Gd QDs were the preferred contrast agents for dual-modality T1-weighted MR imaging and fluorescence imaging with high spatial resolution. Moreover, excellent temporal resolution and high tissue penetration depth were also achieved by fluorescence imaging. These results indicate the potential of Ag2Se-Gd QDs as multifunctional contrast agents for multimodal imaging in clinical diagnosis and research.

Ultrasmall Pb:Ag2 S Quantum Dots with Uniform Particle Size and Bright Tunable Fluorescence in the NIR-II Window.

Ag2 S quantum dots (QDs) are well-known near-infrared fluorophores and have attracted great interest in biomedical labeling and imaging in the past years. However, their photoluminescence efficiency is hard to compete with Cd-, Pb-based QDs. The high Ag+ mobility in Ag2 S crystal, which causes plenty of cation deficiency and crystal defects, may be responsible mainly for the low photoluminescence quantum yield (PLQY) of Ag2 S QDs. Herein, a cation-doping strategy is presented via introducing a certain dosage of transition metal Pb2+ ions into Ag2 S nanocrystals to mitigate this intrinsic shortcoming. The Pb-doped Ag2 S QDs (designated as Pb:Ag2 S QDs) present a renovated crystal structure and significantly enhanced optical performance. Moreover, by simply adjusting the levels of Pb doping in the doped nanocrystals, Pb:Ag2 S QDs with bright emission (PLQY up to 30.2%) from 975 to 1242 nm can be prepared without altering the ultrasmall particle size (≈2.7-2.8 nm). Evidently, this cation-doping strategy facilitates both the renovation of crystal structure of Ag2 S QDs and modulation of their optical properties.

Controllable synthesis of nanocrystals in droplet reactors.

In recent years, a broad range of nanocrystals have been synthesized in droplet-based microfluidic reactors which provide obvious advantages, such as accurate manipulation, better reproducibility and reliable automation. In this review, we initially introduce general concepts of droplet reactors followed by discussions of their main functional regions including droplet generation, mixing of reactants, reaction controlling, in situ monitoring, and reaction quenching. Subsequently, the enhanced mass and heat transport properties are discussed. Next, we focus on research frontiers including sequential multistep synthesis, intelligent synthesis, reliable scale-up synthesis, and interfacial synthesis. Finally, we end with an outlook on droplet reactors, especially highlighting some aspects such as large-scale production, the integrated process of synthesis and post-synthetic treatments, automated droplet reactors with in situ monitoring and optimizing algorithms, and rapidly developing strategies for interfacial synthesis.

Real-Time Dissection of Distinct Dynamin-Dependent Endocytic Routes of Influenza A Virus by Quantum Dot-Based Single-Virus Tracking.

Entry is the first critical step for the infection of influenza A virus and of great significance for the research and development of antiflu drugs. Influenza A virus depends on exploitation of cellular endocytosis to enter its host cells, and its entry behaviors in distinct routes still need further investigation. With the aid of a single-virus tracking technique and quantum dots, we have realized real-time and multicolor visualization of the endocytic process of individual viruses and comprehensive dissection of two distinct dynamin-dependent endocytic pathways of influenza A virus, either dependent on clathrin or not. Based on the sequential progression of protein recruitment and viral motility, we have revealed the asynchronization in the recruitments of clathrin and dynamin during clathrin-dependent entry of the virus, with a large population of events for short-lived recruitments of these two proteins being abortive. In addition, the differentiated durations of dynamin recruitment and responses to inhibitors in these two routes have evidenced somewhat different roles of dynamin. Besides promoting membrane fission in both entry routes, dynamin also participates in the maturation of a clathrin-coated pit in the clathrin-dependent route. Collectively, the current study displays a dynamic and precise image of the entry process of influenza A virus and elucidates the mechanisms of distinct entry routes. This quantum dot-based single-virus tracking technique is proven to be well-suited for investigating the choreographed interactions between virus and cellular proteins.

Near-Infrared Fluorescent Ag2 Se-Cetuximab Nanoprobes for Targeted Imaging and Therapy of Cancer.

Theranostic nanoprobes integrated with diagnostic imaging and therapy capabilities have shown great potential for highly effective tumor therapy by realizing imaging-guided drug delivery and tumor treatment. Developing novel high-performance nanoprobes is an important basis for tumor theranostic application. Here, near-infrared (NIR) fluorescent and low-biotoxicity Ag2 Se quantum dots (QDs) have been coupled with cetuximab, a clinical antiepidermal growth factor receptor antibody drug for tumor therapy, via a facile bioconjugation strategy to prepare multifunctional Ag2 Se-cetuximab nanoprobes. Compared with the Ag2 Se QDs alone, the Ag2 Se-cetuximab nanoprobes display faster and more enrichment at the site of orthotopic tongue cancer, and thus present better NIR fluorescence contrast between the tumor and the surrounding regions. At 24 h postinjection, the NIR fluorescence of Ag2 Se-cetuximab nanoprobes at the tumor site is still easily detectable, whereas no fluorescence is observed for the Ag2 Se QDs. Moreover, the Ag2 Se-cetuximab nanoprobes have also significantly inhibited the tumor growth and improved the survival rate of orthotopic tongue cancer-bearing nude mice from 0% to 57.1%. Taken together, the constructed multifunctional Ag2 Se-cetuximab nanoprobes have achieved combined targeted imaging and therapy of orthotopic tongue cancer, which may greatly contribute to the development of nanotheranostics.

Purification of quantum dot-based bioprobes via high-performance size exclusion chromatography.

Due to excellent optical properties, quantum dots (QDs) have been widely applied to sensing, labeling, and imaging. For the fabrication of QD-based bioprobes, purification is usually the crucial step. Hydrophilic octylamine grafted polyacrylic acid modified QDs (OPA-QDs) were prepared, and purified by high-performance size exclusion chromatography (HPSEC) to remove excess OPA and aggregated QDs. The percentage of suspended agglomerates of OPA-QDs in the unpurified OPA-QDs increases from 4% to 31% through a year, but the purified OPA-QDs of the same batch possess excellent colloidal stability for at least one year. Subsequently, QD-based bioprobes were fabricated by the conjugation between QDs and streptavidin (SA) or antibody (IgG), generating QD-SA and QD-IgG, respectively, which were purified via HPSEC. Finally, the resulting QD-SA and QD-IgG were adopted to detect tumour markers on slices and showed specific positive signals without nonspecific adsorption, which was contrary to the unpurified QD-IgG. Thus, the HPSEC-coupled system proposed in the current work is potent and universal for the generation of purified and monodisperse QD-based bioprobes, which is promising in the nanobiodetection field.

Dissecting the Factors Affecting the Fluorescence Stability of Quantum Dots in Live Cells.

Labeling and imaging of live cells with quantum dots (QDs) has attracted great attention in the biomedical field over the past two decades. Maintenance of the fluorescence of QDs in a biological environment is crucial for performing long-term cell tracking to investigate the proliferation and functional evolution of cells. The cell-penetrating peptide transactivator of transcription (TAT) is a well-studied peptide to efficiently enhance the transmembrane delivery. Here, we used TAT peptide-conjugated QDs (TAT-QDs) as a model system to examine the fluorescence stability of QDs in live cells. By confocal microscopy, we found that TAT-QDs were internalized into cells by endocytosis, and transported into the cytoplasm via the mitochondria, Golgi apparatus, and lysosomes. More importantly, the fluorescence of TAT-QDs in live cells was decreased mainly by cell proliferation, and the low pH value in the lysosomes could also lower the fluorescence intensity of intracellular QDs. Quantitative analysis of the amount of QDs in the extracellular region and whole cells indicated that the exocytosis was not the primary cause of fluorescence decay of intracellular QDs. This work facilitates a better understanding of the fluorescence stability of QDs for cell imaging and long-term tracking in live cells. Also, it provides insights into the utility of TAT for transmembrane transportation, and the preparation and modification of QDs for cell imaging and tracking.

Exploring sialic acid receptors-related infection behavior of avian influenza virus in human bronchial epithelial cells by single-particle tracking.

Human respiratory tract epithelial cells are the portals of human infection with influenza viruses. However, the infection pathway of individual avian influenza viruses in human respiratory cells remains poorly reported so far. The single-particle tracking technique (SPT) is a powerful tool for studying the transport mechanism of biomolecules in live cells. In this work, we use quantum dots to label avian influenza H9N2 virus and elaborate on the infection mechanism of the virus in human bronchial epithelial (HBE) cells using a three-dimensional SPT technique. We have found that the H9N2 virus can infect HBE cells directly and the virus infection follows an actin filament- and microtubule-dependent process with a three-stage pattern. The transport behaviors show a high degree of consistency between the sialic acid receptors and the influenza virus. Real-time SPT provides dynamic evidence of the sialic acid receptors-related infection behavior of the avian influenza virus in live cells. The study of the influence of sialic acid receptors on virus infection may contribute to a better understanding of the cross-species transmission of the avian influenza virus.

Evaluation of nonspecific interactions between quantum dots and proteins.

A method based on the AFM and colloidal probe techniques was proposed to directly measure nonspecific interactions between QDs and different proteins with respective sizes and isoelectric points. Results indicated that van der Waals forces were the leading force, while electrostatic interactions also played an important role in nonspecific interactions.

Fast and high-accuracy localization for three-dimensional single-particle tracking.

We report a non-iterative localization algorithm that utilizes the scaling of a three-dimensional (3D) image in the axial direction and focuses on evaluating the radial symmetry center of the scaled image to achieve the desired single-particle localization. Using this approach, we analyzed simulated 3D particle images by wide-field microscopy and confocal microscopy respectively, and the 3D trajectory of quantum dots (QDs)-labeled influenza virus in live cells. Both applications indicate that the method can achieve 3D single-particle localization with a sub-pixel precision and sub-millisecond computation time. The precision is almost the same as that of the iterative nonlinear least-squares 3D Gaussian fitting method, but with two orders of magnitude higher computation speed. This approach can reduce considerably the time and costs for processing the large volume data of 3D images for 3D single-particle tracking, which is especially suited for 3D high-precision single-particle tracking, 3D single-molecule imaging and even new microscopy techniques.

Mechanism-oriented controllability of intracellular quantum dots formation: the role of glutathione metabolic pathway.

Microbial cells have shown a great potential to biosynthesize inorganic nanoparticles within their orderly regulated intracellular environment. However, very little is known about the mechanism of nanoparticle biosynthesis. Therefore, it is difficult to control intracellular synthesis through the manipulation of biological processes. Here, we present a mechanism-oriented strategy for controlling the biosynthesis of fluorescent CdSe quantum dots (QDs) by means of metabolic engineering in yeast cells. Using genetic techniques, we demonstrated that the glutathione metabolic pathway controls the intracellular CdSe QD formation. Inspired from this mechanism, the controllability of CdSe QD yield was realized through engineering the glutathione metabolism in genetically modified yeast cells. The yeast cells were homogeneously transformed into more efficient cell-factories at the single-cell level, providing a specific way to direct the cellular metabolism toward CdSe QD formation. This work could provide the foundation for the future development of nanomaterial biosynthesis.

Ag2Se quantum dots with tunable emission in the second near-infrared window.

Quantum dots (QDs) with fluorescence in the second near-infrared window (NIR-II, 1000-1400 nm) are ideal fluorophores for in vivo imaging of deep tissue with high signal-to-noise ratios. Ag2Se (bulk band gap 0.15 eV) is a promising candidate for preparing NIR-II QDs. By using 1-octanethiol as ligand to effectively balance the nucleation and growth, tuning the fluorescence of Ag2Se QDs was successfully realized in the NIR-II window ranged from 1080 to 1330 nm. The prepared Ag2Se QDs can be conveniently transferred to the aqueous phase by ligand exchange, showing great potential for multicolor NIR-II fluorescence imaging in vivo.

One-step sensitive detection of Salmonella typhimurium by coupling magnetic capture and fluorescence identification with functional nanospheres.

Sensitive, rapid, and reliable detection of bacteria has always been pursued due to the great threat of the bacteria to human health. In this study, a convenient one-step strategy for detecting Salmonella typhimurium was developed. Immunomagnetic nanospheres (IMNS) and immunofluorescent nanospheres (IFNS) were used to specifically capture and recognize S. typhimurium simultaneously. After magnetic separation, the sandwich immune complexes (IMNS-bacteria-IFNS) were detected under a fluorescence microscope with a detection limit as low as ca. 10 CFU/mL. When they were detected by fluorescence spectrometer, a linear range was exhibited at the concentration from 10(5) to 10(7) CFU/mL with R(2) = 0.9994. Compared with the two-step detection strategy, in which the bacteria were first captured with the IMNS and subsequently identified with the IFNS, this one-step strategy simplified the detection process and improved the sensitivity. Escherichia coli and Shigella flexneri both showed negative results with this method, indicating that this method had excellent selectivity and specificity. Moreover, this method had strong anti-interference ability, and it had been successfully used to detect S. typhimurium in synthetic samples (milk, fetal bovine serum, and urine), showing the potential application in practice.