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Existing nucleic acid and antigen profiling methods for COVID-19 diagnosis fail to simultaneously meet the demands in sensitivity and detection speed, hampering them from being a comprehensive way for epidemic prevention and control. Thus, effective screening of COVID-19 requires a simple, fast, and sensitive method. Here, we report a rapid assay for ultrasensitive and highly specific profiling of COVID-19 associated antigen. The assay is based on a binding-induced DNA assembly on a nanoparticle scaffold that acts by fluorescence translation. By binding two aptamers to a target protein, the protein brings the DNA regions into close proximity, forming closed-loop conformation and resulting in the formation of the fluorescence translator. Using this assay, saliva nucleocapsid protein (N protein) has been profiled quantitatively by converting the N protein molecule information into a fluorescence signal. The fluorescence intensity is enhanced with increasing N protein concentration caused by the metal enhanced fluorescence using a simple, specific, and fast profiling assay within 3 min. On this basis, the assay enables a high recognition ratio and a limit of detection down to 150 fg mL

Natural killer cells (NK cells) are important immune cells which have attracted increasing attention in cancer immunotherapy. Due to the heterogeneity of cells, individual cancer cells show different resistance to NK cytotoxicity, which has been revealed by flow cytometry. Here we used label-free mass cytometry (CyESI-MS) as a new tool to analyze the metabolites in Human Hepatocellular Carcinoma (HepG2) cells at the single-cell level after the interaction with different numbers of NK92 MI cells. A large amount of chemical information from individual HepG2 cells was obtained showing the process of cell apoptosis induced by NK cells. Nineteen metabolites which consecutively change during cell apoptosis were revealed by calculating their average relative intensity. Four metabolic pathways were impacted during cell apoptosis which hit 4 metabolites including glutathione (GSH), creatine, glutamic acid and taurine. We found that the HepG2 cells could be divided into two phenotypes after co-culturing with NK cells according to the bimodal distribution of concentration of these 4 metabolites. The correlation between metabolites and different apoptotic pathways in the early apoptosis cell group was established by the 4 metabolites at the single-cell level. This is a new idea of using single-cell specific metabolites to reveal the metabolic heterogeneity in cell apoptosis which would be a powerful means for evaluating the cytotoxicity of NK cells.

Steroid hormones play important roles in metabolism and metabolic diseases. Currently, various detection methods are employed in clinical labs, mainly immunoassays and LC-MS/MS, but these methods suffer from antibody cross-reactivity or the need for complex LC processes, respectively. Here, we utilized single antibody to capture and separate multiple hormones from samples to avoid LC procedures and used MS/MS to analyze multiple molecules in a single run. In our strategy, testosterone (T), androstenedione (4-AD), and androsterone (ADT) were affinity-captured simultaneously using only T antibody. The qualitative and quantitative analysis of three androgens was realized through MS/MS spectra using testosterone-D

While single-cell mass spectrometry can reveal cellular heterogeneity and the molecular mechanisms of intracellular biochemical reactions, its application is limited by the insufficient detection sensitivity resulting from matrix interference and sample dilution. Herein, we propose an intact living-cell electrolaunching ionization mass spectrometry (ILCEI-MS) method. A capillary emitter with a narrow-bore, constant-inner-diameter ensures that the entire living cell enters the MS ion-transfer tube. Inlet ionization improves sample utilization, and no solvent is required, preventing sample dilution and matrix interference. Based on these features, the detection sensitivity is greatly improved, and the average signal-to-noise (S/N) ratio is about 20 : 1 of single-cell peaks in the TIC of ILCEI-MS. A high detection throughput of 51 cells per min was achieved by ILCEI-MS for the single-cell metabolic profiling of multiple cell lines, and 368 cellular metabolites were identified. Further, more than 4000 primary single cells digested from the fresh multi-organ tissues of mice were detected by ILCEI-MS, demonstrating its applicability and reliability.

NK cell-mediated immunotherapy has received increasing attention in the past decade due to its efficacy and bio-safety. The composition and content of lipids in individual cells are closely related to NK cell-mediated cytotoxicity, especially polyunsaturated fatty acids (PUFA) which are oxidized during NK cell-mediated apoptosis. Here we investigated the changes of lipids in single HepG2 cells by label-free mass cytometry and obtained information on 53 lipids and 13 oxidized lipids after the interaction with NK92 MI cells. We found that the contents of lipids and oxidized lipids of HepG2 cells changed obviously during the NK cell-mediated apoptosis. The HepG2 cells could be classified into two phenotypes after co-culturing with NK92 MI cells based on the ratio of PC(38:6-2OH)/PC(38:6) in individual cells, which may serve as a feature to evaluate NK cell-mediated cytotoxicity. The present work used the lipids and oxidized lipids of individual cells to reveal the heterogeneity in NK cell-mediated apoptosis which would be a powerful method for evaluating the cytotoxicity of NK cells at the single-cell level.

Methylation in the 5′ handle of gRNA disrupts the interaction between the Cas12a and gRNA. The deactivated CRISPR can be reactivated by demethylases, that enabled the development of biosensors and the precise regulation of gene expression.

Disulfide bonds are a class of important post-translational modifications that play important roles in modulating the structures and functions of proteins. Therefore, the mapping of disulfide linkages in peptides and proteins is indispensable for complete structure characterization and functional studies. As disulfide bonds in protonated ions do not dissociate readily under low-energy collision-induced dissociation (CID), they are usually chemically cleaved or activated prior to mass spectrometry (MS) or tandem MS (MS/MS) analysis. In this study, we report a new method that allows the mapping of disulfide linkages in peptides and proteins through meta-chloroperoxybenzoic acid (mCPBA)-based disulfide oxidation and MS/MS. Upon oxidation, the disulfide bond is converted to a thiosulfinate group, i.e., S(═O)-S, in a rapid (>60% yield in 1 min) and highly specific approach in an aqueous phase. The thiosulfinate group is then preferentially cleaved by MS/MS. For interchain disulfide linkages, this leads to a facile peptide chain separation and the identification of disulfide-linked peptides. For intrachain disulfide linkages, collisional activation of the thiosulfinate leads to disulfide cleavage and fragmentation of the peptide backbone constrained by the disulfide loop, enabling a near-complete peptide sequencing. The mCPBA oxidation-based disulfide mapping strategy can be readily integrated with bottom-up or top-down protein analysis for comprehensive protein structure elucidation, e.g., digested lysozyme and intact human insulin.

Metabolites are the end products of cellular vital activities and can reflect the state of cellular to a certain extent. Rapid change of metabolites and the low abundance of signature metabolites cause difficulties in single-cell detection, which is a great challenge in single-cell metabolomics analysis. Mass spectrometry (MS) is a powerful tool that uniquely suited to detect intracellular small-molecule metabolites and has shown good application in single-cell metabolite analysis. In this mini-review, we describe three types of emerging technologies for MS-based single-cell metabolic analysis in recent years, including nano-ESI-MS based single-cell metabolomics analysis, high-throughput analysis via flow cytometry, and cellular metabolic imaging analysis. These techniques provide a large amount of single-cell metabolic data, allowing the potential of MS in single-cell metabolic analysis is gradually being explored and is of great importance in disease and life science research.

Existing structured illumination microscopy (SIM) allows super-resolution live-cell imaging in few color channels that provide merely morphological information but cannot acquire the sample spectrum that is strongly relevant to the underlying physicochemical property. We develop hyperspectral SIM which enables high-speed spectral super-resolution imaging in SIM for the first time. Through optically mapping the three-dimensional (x, y, and λ) datacube of the sample to the detector plane, hyperspectral SIM allows snapshot spectral imaging of the SIM raw image, detecting the sample spectrum while retaining the high-speed and super-resolution characteristics of SIM. We demonstrate hyperspectral SIM imaging and reconstruct a datacube containing 31 super-resolution images of different wavelengths from only 9 exposures, achieving a 15 nm spectral resolution. We show time-lapse hyperspectral SIM imaging that achieves an imaging speed of 2.7 s per datacube-31-fold faster than the existing wavelength scanning strategy. To demonstrate the great prospects for further combining hyperspectral SIM with various spectral analysis methods, we also perform spectral unmixing of the hyperspectral SIM result while imaging the spectrally overlapped sample.

The formation of biomolecular condensates is driven by liquid-liquid phase separation, which is prevalent in cells to govern crucial cellular functions. However, understanding the properties of phase-separated condensates remains very challenging for the lack of suitable techniques. Here, we report a photoluminescence lifetime imaging method for real-time monitoring of phase-separated condensates, both in vitro and in living cells, using a microsecond-scale photoluminescence lifetime probe based on iridium complex. The probe has a large Stokes shift, excellent cell permeability, and minimal cell autofluorescence interference. With this method, the dynamic process of phase separation of fused in sarcoma protein has been well explored, showing high spatiotemporal resolution and high throughput. Beginning with initial formation, the protein droplets get bigger and more viscous, and then a final maturation to solidified aggregates has been characterized. This study paves the path for a deeper understanding of the properties of phase-separated biomolecular condensates.

Single particle tracking (SPT) has long been utilized for investigation of complex system dynamics such as nanoparticle-cell interaction, however, the analysis of individual particle motions is always a difficult issue. Existing methods treat each data point or fragment on the recorded trajectory as an isolated “atom” and determine their relationship based on externally predefined models or physical states, which inevitably lead to oversimplification of the associated spatiotemporal complexity. Herein, inspired by the historical analysis in social science, we propose a modeless preprocessing framework for SPT analysis based on the “history” of the particle. This new strategy consists of 3 steps: (1) assign a “history” to each data point and construct successive overlapped historical vectors; (2) perform unsupervised clustering in the vector space to find their relative differences; (3) project differences back to the trajectory by coloring each point accordingly for visualization. As a result, the inner heterogeneity of the particle motion self-emerges as a colored trajectory, exhibiting a global picture of the local state transitions and providing valuable information for further model-based analysis. Since the complexity issues at various spatiotemporal scales have attracted increasing attention, and individual objects such as single molecules, cells, vehicles and even stars in the universe could all be treated as “single particles”, this presuppositionless data preprocessing approach could help the investigations of many complex systems in fundamental research.

Monitoring clinical biomarkers, such as testosterone in serum, is important for disease assessment. Due to the very low concentration of testosterone in serum, we have developed a new strategy for its enrichment in serum samples by magnetic molecularly imprinted polymers (MMIPs) technology and detection by nano-electrospray ionization mass spectrometry (Nano-ESI-MS). Testosterone was selectively extracted and enriched by the imprinted polymers on the surface of magnetic particles and the complex matrix was eliminated from the serum. The linear calibration curve was in the range of 0.1-10 μg/L and the limit of detection was 11.4 ng/L. The recovery and repeatability of the spiked serum were satisfactory. These results demonstrate that the proposed method is a promising approach for quantitative analysis of testosterone in serum.

A multiplex bacterial assay method that combines S1 nuclease pretreatment and ICP-MS-based elemental labels is presented in this work. Six intestinal related bacteria were identified at the species level and quantified simultaneously without isolation culturing. This method could be extended to assay a mixed bacterial community for point-of-care diagnosis.

Skeletal muscle tissue is composed of various muscle cell types which differ in physiological functions. Changes in cell type composition of skeletal muscle are associated with the development of metabolic diseases. Skeletal muscle cell types are currently distinguished by immunofluorescence (IF) staining based on myosin heavy chain (MHC) isoform difference. However, it remains a challenge to provide metabolic fingerprints of different muscle cell types by IF staining. Therefore, in this study, we proposed a method to examine metabolite distribution within different cell types by time-of-flight secondary ion mass spectrometry (TOF-SIMS) with high spatial resolution. Skeletal muscle samples from C57/BL6 mice were obtained by slicing. Cell types in TOF-SIMS images were labelled corresponding to IF images from the same region of serially cut sections. Mass spectra corresponding to individual muscle cells were extracted to compare metabolic fingerprints among cell types. Skeletal muscle cells were classified into two clusters based on the mass spectra of individual cells. Unsaturated diacylglycerol (DG) and fatty acid (FA) species were found to be distributed in a cell-type dependent manner. Moreover, relative quantification showed that the content of unsaturated DGs, oleic acid and linoleic acid was higher in type I and type IIA cells than in type IIB cells. TOF-SIMS in combination with IF enables us to directly visualize metabolite distribution in different cell types, to find potential biomarkers for cell type classification. TOF-SIMS imaging coupled with IF staining has been proved to be a promising tool for metabolic fingerprinting of different skeletal muscle cell types.

The metal ion-catalyzed (Ru+, Ir+) reactions of ethanol converting to butanol have been studied via ICP-MS/MS. The detection of organic small molecule intermediates (acetaldehyde and butyraldehyde) was achieved in both Ru+/C2H5OH and Ir+/C2H5OH systems. Several metal-adduct intermediates were observed in these reactions, improving the understanding of the reaction mechanism of ethanol transformation. The possible formation pathways of intermediates have been discussed.

Guanosine-based supramolecular hydrogels are particularly of interest for biomaterial and biomedical purposes, as they are generally biocompatible and stimuli-responsive. We found a strong and long-life transparent hydrogel made by mixing guanosine (G) with 1 equiv. of 2-formylbenzeneboronic acid (2FPB) and KOH. Alkali cations can assist the stacking of individual G-quartet to give extended nanowires, but only K+ ion induces the formation of a stable and self-supporting network hydrogel for a couple of months. Data from variable temperature NMR indicated that guanosine 2-formylbenzeneborate ester and G are the key components of the self-assembly. Further, G-2FPB-K+ hydrogel solution can induce berberine (BBR) fluorescence, showing high selectivity to K+ ion and anti-ion interference capability. A good linear relationship between fluorescent intensity and K+ concentration allowed us to directly detect K+ levels in human blood serum.

The selective measurement of cellular pH at specific regions is significant for the in-depth study of cell functions and pathological processes. Herein, we combined our previously developed rhodamine B-labeled cell-penetrating peptide spirolactam derivative with quantum dots to create a ratiometric nanobiosensor for the simultaneous sensing and imaging of lysosomes and extracellular pH.

Urban planning requires more public involvement and larger group participation to achieve scientific and democratic decision making. Crowdsourcing is a novel approach to gathering information, encouraging innovation and facilitating group decision-making. Unfortunately, although previous research has explored the utility of crowdsourcing applied to urban planning theoretically, there are still rare real practices or empirical studies using practical data. This study aims to identify the prospects for implementing crowdsourcing in urban planning through a bibliometric analysis on current research. First, database and keyword lists based on peer-reviewed journal articles were developed. Second, semantic analysis is applied to quantify co-occurrence frequencies of various terms in the articles based on the keyword lists, and in turn a semantic network is built. Then, cluster analysis was conducted to identify major and correlated research topics, and bursting key terms were analyzed and explained chronologically. Lastly, future research and practical trends were discussed. The major contribution of this study is identifying crowdsourcing as a novel urban planning method, which can strengthen government capacities by involving public participation, i.e., turning governments into task givers. Regarding future patterns, the application of crowdsourcing in urban planning is expected to expand to transportation, public health and environmental issues. It is also indicated that the use of crowdsourcing requires governments to adjust urban planning mechanisms.

α-Synuclein is a central player in Parkinson's disease (PD) pathology. Various point mutations in α-synuclein have been identified to alter the protein-phospholipid binding behavior and cause PD. Therefore, exploration of α-synuclein-phospholipid interaction is important for understanding the PD pathogenesis and helping the early diagnosis of PD. Herein, a phospholipid-decorated liquid crystal (LC)-aqueous interface is constructed to investigate the binding between α-synucleins (wild-type and six familial mutant A30P, E46K, H50Q, G51D, A53E and A53T) and phospholipid. The application of deep learning analyzes and reveals distinct LC signatures generated by the binding of α-synuclein and phospholipid. This system allows for the identification of single point mutant α-synucleins with an average accuracy of 98.3±1.3% in a fast and efficient manner. We propose that this analytical methodology provides a new platform to understand α-synuclein-lipid interactions, and can be potentially developed for easy identification of α-synuclein mutations in common clinic.

Disulfide bonds are a class of important post-translational modifications that play important roles in modulating the structures and functions of proteins. Therefore, the mapping of disulfide linkages in peptides and proteins is indispensable for complete structure characterization and functional studies. As disulfide bonds in protonated ions do not dissociate readily under low-energy collision-induced dissociation (CID), they are usually chemically cleaved or activated prior to mass spectrometry (MS) or tandem MS (MS/MS) analysis. In this study, we report a new method that allows the mapping of disulfide linkages in peptides and proteins through

The tool box of site-specific cleavage for nucleic acid has been an increasingly attractive subject. Especially, the recent emergence of the orthogonally activatable DNA device is closely related to the site-specific scission. However, most of these cleavage strategies are based on exogenous assistance, such as laser irradiation. Endogenous strategies are highly desirable for the orthogonally regulatable DNA machine to explore the crucial intracellular biological process and cell signal network. Here, we found that the accurate site-specific cleavage reaction of phosphorothioate (PT) modified DNA by using myeloperoxidase (MPO). A scissors-like mechanism by which MPO breaks PT modification through chloride oxidation has been revealed. Furthermore, we have successfully applied the scissors to activate PT-modified hairpin-DNA machines to produce horseradish peroxidase (HRP)-mimicking DNAzyme or initiate hybridization chain reaction (HCR) amplification. Since MPO plays an important role in the pathway related to oxidative stress in cells, through the HCR amplification activated by this tool box, the oxidative stress in living cells has been robustly imaged. This work proposes an accurate and endogenous site-specific cleavage tool for the research of biostimuli and the construction of DNA molecular devices.

Discriminating various leukocyte subsets with specific functions is critical due to their important roles in the development of many diseases. Here, we proposed a general strategy to unravel leukocytes heterogeneity and screen differentiated metabolites as biomarker candidates for leukocyte subtypes using the label-free mass cytometry (CyESI-MS) combined with a homemade data processing workflow. Taking leukemia cells as an example, metabolic fingerprints of single leukemia cells were obtained from 472 HL-60, 416 THP-1, 313 U937, 356 Jurkat, and 366 Ramos cells, with throughput up to 40 cells/min. Five leukemia subtypes were clearly distinguished by unsupervised learning t-SNE analysis of the single-cell metabolic fingerprints. Cell discrimination in the mixed leukemia cell samples was also realized by supervised learning of the single-cell metabolic fingerprints with high recovery and good repetition (98.31 ± 0.24%, -102.35 ± 4.82%). Statistical analysis and metabolite assignment were carried out to screen characteristic metabolites for discrimination and 36 metabolites with significant differences were annotated. Then, differentiated metabolites for pairwise discrimination of five leukemia subtypes were further selected as biomarker candidates. Furthermore, discriminating cultured leukemia cells from human normal leukocytes, separated from fresh human peripheral blood, was performed based on single-cell metabolic fingerprints as well as the proposed biomarker candidates, unveiling the potential of this strategy in clinical research. This work makes efforts to realize high-throughput single-leukocyte metabolic analysis and metabolite-based discrimination of leukocytes. It is expected to be a powerful means for the clinical molecular diagnosis of hematological diseases.

Understanding the spatiotemporal dynamics of particles in a complex biological environment is crucial for the study of related biological processes. To analyze the complicated trajectories recorded from single-particle tracking (SPT), we have proposed a method named SEES based on historical experience vector analysis, which allows both the global patterns and local state continuities of a trajectory to emerge by themselves as color segments without predefined models. This method implements a data-driven strategy and thus uncovers the hidden information with less prior knowledge or subjective bias. Here, we demonstrate its efficiency by comparing its performance with the Hidden Markov model (HMM), one of the most widely used methods in time series processing. The results demonstrated that the SEES operator was more sensitive in identifying rare events and could utilize multivariable observations in the dynamic processes to uncover more details. We applied the method to analyze the dynamics of nanoparticles interacting with live cells expressing programmed death ligand 1 (PD-L1) on the membrane. The results showed that the SEES operator can successfully pinpoint the transmembrane rare events, visualize the on-membrane "Brownian searching" motion, and evaluate different dynamics among multiple trajectories. Furthermore, we found that the PD-L1 expression level on the cell membrane affected the rotation behavior of the nanoparticle as well as the cellular uptake efficiency. These findings enabled by SEES could potentially help the rational design of highly efficient nanocargoes.

Lipids exert substantial influences on vertebrate embryogenesis, but their metabolic dynamics at detailed structural levels remains elusive, primarily owing to the lack of a tool capable of resolving their huge structural diversity. Herein, we present the first large-scale and spatiotemporal monitoring of unsaturated lipids with C=C specificity in single developing zebrafish embryos enabled by photochemical derivatization and tandem mass spectrometry (MS). The lipid isomer composition was found extremely stable in yolk throughout embryogenesis, while notable differences in ratios of C=C location (e.g., PC 16:0_16:1 (7) vs. 16:0_16:1 (9)) and fatty acyl composition isomers (e.g., PC 16:1_18:1 vs. 16:0_18:2) were unveiled between blastomeres and yolk from zygote to 4 h post fertilization (hpf). From 24 hpf onwards, lipid isomer compositions in embryo head and tail evolved distinctively with development, suggesting a meticulously regulated lipid remodeling essential for cell division and differentiation. This work has laid the foundation for functional studies of structurally defined lipids in vertebrate embryology.

5-Carboxylfluorescein (FAM) is a conventional pH-responsive fluorophore widely used in fluorescence labeling and imaging. Because of its nonfluorescent structure under acidic conditions, FAM has long been limited to pH determination in a neutral–basic environment. Here, we modified the optical properties of FAM with cationic arginine-rich cell-penetrating peptides (CPPs), tuning the pKa value of FAM to adapt well to pH measurement under diverse pH conditions. With increasing length of polyarginine, the pKa value of FAM was tuned from 6.20 ± 0.06 to 5.17 ± 0.05. The key mechanism for pKa variations was attributed to intramolecular electrostatic attraction and the positive charge of cationic CPPs tend to stabilize the fluorescent dianionic form of FAM. Apart from tunable pKa, arginine-rich CPPs also improved the water solubility, membrane permeability, and organelle-specific localization of FAM. Two conjugated probes FAM-R12 and FAM-(Fxr)3 were selected to monitor intracellular pH fluctuations. Compared to ...

Fluorescent carbon nanodots (CNDs) capable of dual emission blue and red light (B-R-CNDs) (460 and 610 nm) have been synthesized by a simple and low cost electrochemical method of discharging indol...

Changes of metabolite concentrations in single cells are significant for exploring the dynamic regulation of important biological processes, such as cell development and differentiation. Accurate quantitation of metabolites is essential for single cell analysis. In this work, we proposed a quantitative method for single-cell metabolites by combining microwell array with droplet microextraction-mass spectrometry. The microwell can confine both single cells and extraction solvent in defined space, avoiding the irregular spread of trace internal standard solution during microextraction, which was the key to improve the precision and accuracy of quantification in extremely small-volume single-cell samples. Glucose-phosphate as a crucial metabolite in glycolysis was detected and quantified in single cells at this work. The calibration curve of glucose-phosphate was obtained with a linear range from amol (10–18 mol) to fmol (10–15 mol), providing the foundation of metabolite quantitation of single cells. We app...

Chemical warfare agents (CWAs) are highly toxic compounds that have been used in wars or terrorist attacks. There is an urgent need to develop fast, inexpensive and sensitive on-site CWA inspection techniques. In this study, a low-temperature plasma (LTP) probe was directly coupled with a metal needle tip where sample molecules were desorbed and ionized in the ambient environment for subsequent analysis using mass spectrometry (MS). We found that metal needles greatly increased the ionization efficiencies compared with non-conductive sample substrates. Based on this finding, a method for rapid and highly sensitive detection and quantification of CWA-related compounds, including dimethyl methylphosphonate (DMMP), isopropyl methylphosphonic acid (IMPA), methylphosphonic acid (MPA) and thiodiglycol (TDG) was established. The whole workflow was completed in 10 s with a dynamic range of 1–500 μg L−1, and the limit of detection (LOD) was 500–980 ng L−1. Analytes were spiked in real-world water samples and quantified using this approach with satisfactory recoveries. Our results indicated that metal needle-enhanced LTP-MS will be a useful technique for the highly sensitive detection of CWAs.

Ratiometric quantification for competitive immunoassay based on internal standard element detection utilizing inductively coupled plasma mass spectrometry (ICP-MS) as multiplex readout has been demonstrated. The Beta-2-microglobulin (β2-MG) associated with clinical diseases was detected by Y-labeled capture antibody used as internal standard probes and Sm-labeled antigen used as report probes via antigen-antibody reaction. The ratiometric quantification was achieved by taking the signal ratio of Sm/Y. The ratiometric method could compensate for the particle loss and suppress the signal fluctuation, which improved the precision of the quantitative result. Under the optimized conditions, the calibration curves for β2-MG antigen was linear in the range of 0.25–8.0 μg/mL with a detection limit of 0.17 μg/mL (3σ, n = 11). The recoveries of 96.5%–132% were obtained for serum samples spiked with different concentration standards, and the relative standard deviation (RSD) was less than 10%. The β2-MG results in serum samples obtained by the proposed method correlated well with those obtained by time-resolved fluorescence immunoassay (r = 0.947). This proposed method provides a simpler labeling strategy for ratiometric quantification of immunoassay.

An efficient synthetic method of tetracyclic 3,4-fused indoles and dihydroindoles via rhodium-catalyzed (3+2) cycloaddition of N-tosyl-4-(2-phenoxyphenyl)-1,2,3-triazole was described. The aromatized xanthene derivatives can be achieved in a one-pot synthesis starting from 1-ethynyl-2-phenoxybenzene. The xanthene-based fused heterocycles were considered as the valuable fluorophore.

A simple multiplexed digital microRNA detection strategy with fluorescence flow cytometry was proposed. By isothermal ligation-rolling circle amplification, multiplexed microRNAs could be simultaneously converted to a series of nanoflower balls (NFBs) and counted by flow cytometry directly.

Objectives In this study, a new immunoassay for the simultaneous determination of pepsinogen I (PGI) and pepsinogen II (PGII) in serum based on element labeling strategy coupled with inductively coupled plasma mass spectrometry (ICP‐MS) detection was proposed. Methods The sandwich‐type immunoassay was used to simultaneously detect PGI and PGII in serum. PGI and PGII were captured by anti‐PGI and anti‐PGII antibody immobilized on the magnetic beads and then banded with Eu3+ labeled anti‐PGI detection antibody and Sm3+ labeled anti‐PGII detection antibody, followed by ICP‐MS detection. Results The linear correlation coefficient (R 2) of PGI and PGII standard curves was .9938 and .9911, with the dynamic range of 0‐200 ng/mL and 0‐60 ng/mL, respectively. The limit of detection for PGI and PGII was 1.8 ng/mL and 0.3 ng/mL, respectively. The average recovery was 101.41% ± 6.74% for PGI and 101.47% ± 4.20% for PGII. Good correlations were obtained between the proposed method and CLIA (r = .9588 for PGI, r = .9853 for PGII). Conclusions We established a mass spectrometry‐based immunoassay for the simultaneous detection of PGI and PGII in a single analysis. The element tagged immunoassay coupled with ICP‐MS detection has high sensitivity, accuracy, and specificity in clinical serum sample analysis.

Developing rapid and reliable method for simultaneous hormones quantitation is of great significant because of important roles of hormones in metabolism. However, current methods are faced with problems of low throughput or complicated operation procedure to remove matrices from serum samples in routine clinical diagnosis. In the present work, a multilayer PS-MS method was developed for rapid and simple detection of hormones. In the strategy, multilayer filter paper acted as the Liquid Chromatography in LC-MS/MS for separation of hormones and biological matrices. Qualitative and quantitative analysis of three hormones, testosterone (T), androsterone (ADT) and androstenedione (4-AD) were realized through MS/MS spectra. The method exhibited linearity in the range of 0.02–2 μg/L and the results of recovery and repeatability were satisfactory for standard samples and spiked serum. The time-cost of a whole detection process was less than 3 min. The established multilayer PS-MS realized rapid, simple and reliable quantitative analysis of various hormones and provided broad prospect for clinical analysis of small molecules in different biological samples. Moreover, it provides a novel MS approach with high through-put and free HPLC, meeting the requirements of point-of-care testing (POCT).

High-throughput sample preparation is important in clinical mass spectrometry (MS) analysis. In this work, we developed a method by coupling Nano-ESI-MS with magnetic SPE using a MOF material, Fe3O4@UiO-66, as sorbent for simultaneous determination of testosterone (T) and androstenedione (4-AD) in serum samples. The proposed method presented good linearity in the range of 0.1–5 μg/L and the recoveries of serum samples were 90.7%-110.0% and 101.7%-108.7% for T and 4-AD, respectively. The results indicated that the Fe3O4@UiO-66 is an ideal sorbent for androgens extraction and the method is reliable for high-throughput quantitation of T and 4-AD in clinical applications.

A new instrument based on VUV laser desorption/ionization (VUVDI) method was used to measure the mass spectrum of the cholesterol standard and the mass spectrometry images (MSI) of intact cholesterol in a mouse esophagus tissue section and mouse zygotes. The VUVDI mass spectrum of the cholesterol standard shows dominant peaks from the parent molecules. In the case of the mouse esophagus tissue section, the ion images with a lateral resolution of ∼ 4 μm reveal that the cholesterol populates mainly in the mucosae. The experimental results indicate that the VUVDI method may provide a new MSI tool to measure the intact cholesterol in tissues and cells.

Mass spectrometry imaging (MSI) is a crucial label-free method to distinguish the localization patterns in single cells. MALDI-TOF MS and ToF-SIMS are now bearing the responsibility. However, MALDI-TOF MS is limited to micron spatial resolution and ToF-SIMS suffers from severe molecular fragmentation. Here, we proposed a new MSI methodology of vacuum ultraviolet laser desorption/ionization (VUVDI) with high spatial resolution, achieving higher ion yields and less fragmentation compared with ToF-SIMS at submicron level. The fluorescence image and mass spectrum of VUVDI were obtained simultaneously. In addition, the adjustable laser fluence acquired selective detection for different molecular and fragmental ions, thus realizing molecular identification. Furthermore, MSIs of single cells with submicron craters were presented. These results suggest VUVDI is a potential mass spectrometry method that provides a soft ionization source and submicron spatial resolution for molecular analysis in life science.

While studies of collective cell migration and bacteria swarming have tremendously promoted our fundamental knowledge of the complex systematic phenomena, the quantitative characterization of the collective organelles movement at subcellular level is yet to be fully explored. Here we tagged the lysosomes in live cells with fluorescent probe and imaged their spatial motion with wide field microscopy. To quantitatively characterize the collective lysosomal behavior with high spatiotemporal heterogeneity dynamics, we developed the particle collective analysis (PECAN) method based on the single particle tracking techniques. Thousands of trajectories were detected and analyzed in each single cell. The reliability was validated by comparing with traditional PIV method, simulated and experimental data sets. We show that the lysosomes in live cells move collectively with spatial heterogeneous and temporal long-term correlated dynamics. Furthermore, the continuous wavelet analysis suggested the existence of collective lysosomal oscillation in mouse neural cells. Generally, our method provides a practical workflow for characterizing the collective lysosomal motions which can benefit related areas such as organelles mediated drug delivery and cell activity profiling.

Unfortunately, in the original publication of the article, the voucher number in the second paragraph was published erroneously as “YNUSM20160817008”. The corrected voucher number should read as “YNUST201406180002”.

In this work, we demonstrate a novel design that allows rapid online identification and quantitation of unsaturated fatty acid C═C location isomers via epoxidation and ambient mass spectrometry (MS). Unsaturated fatty acid solution was loaded on a paper strip placed between a low-temperature plasma probe and the inlet of a mass spectrometer. Reactive oxygen species in the plasma promoted epoxidation at the C═C, and the product was simultaneously ionized. Upon collision-induced dissociation (CID), the epoxidation product was fragmented to release diagnostic ions specific to the C═C location. The whole analytical workflow can be completed within 5 s and is particularly promising for point-of-care (POC) clinical diagnosis, considering its fast, high-throughput nature, and coupling with paper-based analytical devices.

A novel platform for rapid screening of gaseous catalysts in methane activation using ICP-QQQ-MS was developed. The gaseous ion catalysts could be produced in an ICP torch from the bulk solution. The dynamic reaction cell in the ICP-QQQ-MS could be used as the reactor to mix methane with the gaseous ion catalyst to examine the important parts of the catalytic reaction. In this work, 37 kinds of gaseous catalysts were rapidly screened and the results obtained by using the ICP-QQQ-MS were also validated by using another dielectric barrier discharge reactor. The ICP-QQQ-MS platform was not only suitable for catalyst screening in methane activation, but also suitable for other full reactions.

The identification of trace components from biological media can require derivatization under mild conditions for successful analysis by mass spectrometry (MS). When aqueous droplets (ca. 500 nL) containing a sugar and an amine as reagents are allowed to evaporate they may form long-lasting microthin films in which derivatization reactions can occur fast relative to reaction rates in bulk solution. Evidence is presented that these reactions are heterogeneous in nature and comparisons are made with reactions in pastes and in neat reagent mixtures. Moreover, these thin film reactors can be made stable for the long periods of time that may be necessary to give high product yields. The situation is typified by imine formation from reducing sugars which have reaction times of much more than 1 hour provided that small concentrations (e.g. 20 ppm) of nonvolatile solvents are included. After evaporation of almost all the water, the reaction occurs at an approximately constant rate for the first hour. The rate is two orders of magnitude faster than the reaction in the corresponding homogeneous saturated bulk solution. Conversion of the reagent to the Schiff base product is 67% to 96% efficient in these long-lasting thin films, in sharp contrast to the corresponding derivatization efficiencies in the bulk of less than 1%. This method was used to chemically derivatize and thus to identify, using tandem mass spectrometry, 29 reducing sugars in ca. 1 nL of intracellular fluid from a single onion epidermis cell. A formal description of the kinetics of reversible and irreversible second order reactions in thin films is provided. The effects of thermodynamic and kinetic factors are separated and the measured apparent acceleration factor is shown to represent the ratio of intrinsic rate constants for the microthin film reactor relative to the bulk reaction.

A novel photoluminescence lifetime probe (Ir-TB) has been developed for the detection and imaging of hCE2 in living cells. A large lifetime increase by around 300 ns after the enzymatic reaction makes it an ideal tool to distinguish hCE2-hydrolyzed probes from those non-hydrolyzed ones via PLIM for the first time.

Designing probes for real-time imaging of dynamic processes in living cells is a continuous challenge. Herein, a novel near-infrared (NIR) photoluminescence probe having a long lifetime was exploited for photoluminescence lifetime imaging (PLIM) using an iridium-alkyne complex. This probe offers the benefits of deep-red to NIR emission, a long Stokes shift, excellent cell penetration, low cytotoxicity, and good resistance to photobleaching. This example is the first PLIM probe applicable to the click reaction of copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with remarkable lifetime shifts of 414 ns, before and after click reaction. The approach fully eliminates the background interference and distinguishes the reacted probes from the unreacted probes, thus enabling the wash-free imaging of the newly synthesized proteins within single living cells. Based on the unique properties of the iridium complexes, it is anticipated to have applications for imaging other processes within living cells.

Cell growth and division are important processes for life. To understand the distribution and behaviors of the key molecules that dominate the cell growth and division still remains a challenge. Herein, we propose a strategy for visualization of newly synthesized proteins by TOF-SIMS coupled with metabolic incorporation of 15N labelled amino acids during the cell growth. Moreover, we simultaneously image phosphatidylcholine and cholesterol during the cell division, since TOF-SIMS is especially suitable for this kind of molecules. With the proposed strategy, 2-dimension and 3-dimension images of proteins and lipids are obtained during cell division. We found that phosphatidylcholine was widely distributed in the cleavage furrow while the newly synthesized proteins exist few in this region. This technique will be a valuable tool to study the molecular behaviors and their spatial distribution in single cell.

A novel nanoparticles-quantum dots-based fluorescence sensor array has been developed for sensing proteins and cancer cells. In this study, six types of nanoparticles (NPs, including CuO, ZnO, Eu 2 O 3 , AuNPs, AgNPs, Au-Ag core-shell） conjugated with CdSe quantum dots (QDs) were used as sensing elements to create the multi-sensors of array. The fluorescence of quantum dots was efficiently quenched by nanoparticles. Fluorescence turn-on or further quenching could be observed due to the disruption of the nanoparticles-QDs interaction by proteins, affording distinct fluorescence response patterns. Linear discriminant analysis (LDA) was used to analyze the patterns. Moreover, protein quantification could be performed according to the fluorescence intensity on a specific or single sensing element. The linear dynamic ranges of the sensor array for proteins was in the range of 2–50 μM, and the limits of detection (LODs) was below 2 μM that varied for different proteins. Six cancer cells were also successfully differentiated by their fluorescence response patterns. This work indicates that the nanoparticles-QDs-based sensor array has a great potentials to be used in such fields as proteomics and medical diagnostics.

We constructed a DNA tetrahedron based multicolor nanoprobe, which could simultaneously imaging of three tumor-related mRNAs in living cells through fluorescence restoration caused by competitive chain replacement reaction. The oligonucleotides used to construct the tetrahedron were extended by adding three 21-base recognition sequences modified with different fluorophores (FAM, Cy3, and Cy5) in the 5' end. Three 11-base complementary sequences modified with quencher (BHQ1 for FAM and BHQ2 for Cy3 and Cy5) were hybridized with the recognition sequences to quench the fluorescence. In the presence of the specific mRNA targets, the recognition sequences hybridized with the targets to form longer duplexes and the fluorescence was restored. Compared with previously reported nanoprobes based on DNA tetrahedron, the multicolor nanoprobe can effectively avoid false positive results.