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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

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

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.

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.

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.

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.

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.

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.

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.

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 ...

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.

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.

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.

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.

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.

We developed a rapid high-throughput screening platform using a modified ICP-MS/MS system for monovalence metal ions catalysts discovery in azide–alkyne cycloaddition. Among the ten monovalence metal ions in the first row of periodic table containing Sc+, Ti+, V+, Cr+, Mn+, Fe+, Co+, Ni+, Cu+, and Zn+, five monovalence metal ions of Sc+, Co+, Ni+, Cu+ and Zn+ ions show relatively stronger catalytic activities than others. The catalytic mechanism of Sc+, Co+ and Ni+ ions is similar to the Cu+ ions, but Zn+ ions take a different catalytic route. A yields range of 77–98% for azide-alkyne cycloaddition was achieved with Sc+, Co+, Ni+, Cu+ and Zn+ ions as catalyst, respectively by using a UV laser ablation reactor in 20 min, notable that the yields of Co+ and Sc+ ions were even higher than the common catalyst of Cu+ ions. The proposed platform would be used not only for catalyst discovery in azide-alkyne cycloaddition, but also for the discovery of single atom/ion catalysts in other organic reactions.

Single nucleotide polymorphisms (SNPs) are the most fundamental internal causes for many genetic diseases. However, the location information on SNPs in a specific DNA sequence is not well acquired through current SNPs detection methods, except for accurate DNA sequencing. Here we report a fluorescence enhancement phenomenon in the process of two silver nanoclusters (AgNCs) approaching closely to form a nanocluster dimer (NCD). The fluorescence intensity is sensitive to the distance between two AgNCs; therefore, the NCD lights into different fluorescence intensities upon binding SNPs targets with mismatched bases at different positions. Interestingly, the fluorescence intensities of the NCD decrease linearly when the position of single mismatched base moves gradually from the middle point to the end of the target DNA. The NCD is a single probe acting as a universal platform to pinpoint various SNP positions. With this single probe, we cannot only identify the existence of SNPs but also pinpoint the location of a specific single mismatched base in the adjacent positions. This strategy is feasible to detect specific gene point mutations in clinical samples.

A colorless and nonfluorescent spirolactam derivative, RhB-R12K, was synthesized by amide condensation between the carboxyl group of rhodamine B (RhB) and the amino group of cell-penetrating peptide (CPP). The fluorescence intensity of RhB-R12K sharply increased as the pH value decreased from 8.0 to 4.9, demonstrating sensitive and reversible response to intracellular pH distribution. This CPP probe was completely water soluble, had low cytotoxicity, was membrane permeable, and was suitable for pH measurement in various organelles by choosing organelle-specific CPP sequences. Interestingly, CPPs acted not only as carriers but also as indispensable parts of fluorophores here. The presence of active groups on the peptides potentially allows for modification with additional dyes to construct multifunctional and ratiometric probes for cell imaging.

Plasma-based ambient mass spectrometry (AMS) exhibits great potential in the direct analysis of raw samples with minimum pretreatment. A variety of plasma-based ionization sources have emerged and presented favorable advantages of their high sensitivity, fast analysis speed, and promising qualitative and quantitative abilities for a wide range of applications. Successive efforts have been made to further improve the plasma-based sources for reliable practical applications. Herein, the instrumentation, mechanisms, novel configurations, and applications of versatile plasma-based AMS techniques have been overviewed, and further perspectives have been discussed in the end.

Some anticarcinogen could treat cancers through regulating the synthesis and degradation of proteins. Therefore, the rules of newly synthesized proteins would be used to evaluate the effect of anticarcinogen. In this study, we coupled noncanonical amino acid tagging with click reaction to label the newly synthesized proteins in normal cells and cancerous cells. We studied the influence of six different drugs on the protein synthesis. The results showed the differences of protein synthesis rate and spatial distribution in normal cells and cancerous cells, as well as their different responding to anticarcinogen stimulation. This approach could help us to understand the growth of proteins and distinguish cancerous cells from normal cells, which would benefit the diagnosis and monitoring of cancer, as well as evaluating the curative effect of drugs. Based on this strategy, more significant biological process about newly synthesized biomolecules would be studied in-depth.

ICP-MS-based multiplex immunoassays have the advantages of low sample consumption and minimized repetitions of tedious procedures. However, this technique is seldom used in clinical tests because of the complicated matrix effect in clinical samples and the discrimination effect to the target proteins. In this work, we developed an ICP-MS-based magnetic immunoassay by using magnetic beads as highly efficient affinity probes and optimizing the magnetic immunoassay parameters. We applied this method to the simultaneous determination of three crucial gynecological tumor biomarkers that are vital in the prognosis and monitoring of the recurrence of breast cancer and ovarian cancer. The measurable ranges of HER-2, HE4 and CA15-3 were 12–1000 ng mL−1, 8–2000 pmol L−1 and 5–200 U mL−1, with detection limits (3σ) of 3.94 ng mL−1, 2.59 pmol L−1 and 1.62 U mL−1, respectively. The relative standard deviations (RSDs) were 2.61% for HER-2 at 15 ng mL−1, 4.90% for HE4 at 25 pmol L−1 and 6.68% for CA15-3 at 10 U mL−1, indicating good accuracy of the method. The spiking test indicated that the present method had a low matrix effect. Under optimized conditions, the simultaneous detection of HER-2, HE4, and CA15-3 in clinical serum samples was performed and the values correlated well with those obtained by the time resolved fluorescence immunoassay (r2 = 0.9568 for HER-2, 0.9485 for HE4, 0.9133 for CA15-3). The present work shows that the ICP-MS-based magnetic immunoassay meets the requirements for the quantification of three crucial gynecological tumor biomarkers in real clinical tests.

Using matrix to enhance the molecular ion signals for biomolecule identification without loss of spatial resolution caused by matrix crystallization is a great challenge for the application of TOF-SIMS in real-world biological research. In this report, graphene oxide (GO) was used as a matrix for TOF-SIMS to improve the secondary ion yields of intact molecular ions ([M + H]+). Identifying and distinguishing the molecular ions of lipids (m/z >700) therefore became straightforward. The spatial resolution of TOF-SIMS imaging could also be improved as GO can form a homogeneous layer of matrix instead of crystalline domain, which prevents high spatial resolution in TOF-SIMS imaging. Lipid mapping in presence of GO revealed the delicate morphology and distribution of single vesicles with a diameter of 800 nm. On GO matrix, the vesicles with similar shape but different chemical composition could be distinguished using molecular ions. This novel matrix holds potentials in such applications as the analysis and imaging of complex biological samples by TOF-SIMS.

Multiplex biomolecular analysis with inductively coupled plasma mass spectrometry (ICP-MS) becomes increasingly important in clinical diagnosis and single cell analysis. However, the sensitivity of ICP-MS-based immunoassay is only comparable or lower than those of fluorescence methods at the present stage. Therefore, designing elemental tags with a large number of metal atoms is necessary to achieve high-sensitive detection. In this work, we proposed a new strategy to build up elemental tag loading with hundreds of rare earth ions by coupling alkyne-DNA chains with rare earth element (REE)-DOTA complexes a click chemistry reaction. There are about 2 orders of magnitude improvement in sensitivity compared with single metal-ion tags. DNA chains with multialkynyl groups were facilely prepared by PCR synthesis. Moreover, the DNA-based elemental tags own excellent water-solubility and biocompatibility. The tags would be potentially applied to mass cytometry and clinical diagnosis.

Hypoxia is a common characteristic of solid tumors, which is caused by the imbalance of oxygen supply and consumption. As the expression level of nitroreductase (NTR) increases in hypoxic solid tumors, NTR is one of the common biomarkers of hypoxia and widely used to evaluate the degree of tumor hypoxia. In this study, we designed and synthesized a highly water-soluble chemiluminescent probe, CL-NTR, for the detection of NTR activity in hypoxic tumors. It was found that the probe could be used to detect NTR with high sensitivity, and the total light photons increased tremendously with 6000-fold after the probe was treated with NTR. The chemiluminescence total light photons emission was directly proportional to the concentration of nitroreductase in the range of 3-55 ng/mL, with a detection limit of 0.947 ng/mL. Finally, the probe was successfully used to evaluate NTR activity in living mice by chemiluminescent imaging. In general, this probe has a remarkable response to NTR, which provides a promising method for the determination of NTR activity in vivo.

Intermediates in the gas-phase reaction of diatomic [TaO]+ with CH4 to generate ethane and ethylene have been explored using ICP-MS/MS complemented by high-level quantum chemical calculations. [TaOH]+, [TaOCH2]+, [TaOC2H6]+and [TaOC2H4]+ were identified and the formation pathways of them were discussed, revealing the mechanistic scenario for this oxidative coupling of methane to the C2 hydrocarbons process.

Current detection methods for paper-based analytical devices (PADs) rely on spectroscopic and electrochemical properties, which place special requirements on the analyte or need analyte labeling. Here, ion-transmission mass spectrometry (MS) was proposed for coupling with PADs to enable rapid in situ MS analysis of the sample on paper. The sample was analyzed directly on paper via analyte ionization by ions transmitted through the paper, generated by a low-temperature plasma probe. Prior to MS analysis, the sample can be separated by paper electrophoresis or by paper chromatography, among a variety of other features offered by PADs. The versatility of this technique was demonstrated by MS analysis of a paper microarray, a mixture of amino acids, and whole blood doped with drugs on PADs.

The salt-induced aggregation, nanoparticle regrowth and self-assembly behaviors of gold nanoparticles (AuNPs) and DNA conjugates could be changed after interaction with different proteins, generating various color changes and a unique fingerprint pattern for each protein. The triple-channel colorimetric signals have been employed for protein discrimination with the naked eye.

Background: Element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis for multiplex detection. However, a further study referring to the standard evaluation and clinical sample verification is needed to ensure its reliability for simultaneous analysis in clinical laboratories. Methods: Carcinoembryonic antigen (CEA) and &alpha, fetoprotein (AFP) were chosen for the duplex immunoassay. The performance of the assay was evaluated according to guidelines from the Clinical and Laboratory Standards Institute (CLSI). Moreover, reference intervals (RIs) of CEA and AFP were established. At last, 329 clinical samples were analyzed by the proposed method and results were compared with those obtained with electrochemiluminescent immunoassay (ECLIA) method. Results: The measurement range of the assay was 2&ndash, 940 ng/mL for CEA and 1.5&ndash, 1000 ng/mL for AFP, with a detection limit of 0.94 ng/mL and 0.34 ng/mL, respectively. The inter-assay and intra-assay imprecision were all less than 6.58% and 10.62%, respectively. The RI of CEA and AFP was 0&ndash, 3.84 ng/mL and 0&ndash, 9.94 ng/mL, respectively. Regarding to clinical sample detection, no significant difference was observed between the proposed duplex assay and the ECLIA method. Conclusions: The ICP-MS-based duplex immunoassay was successfully developed and the analytical performance fully proved clinical applicability. Well, this could be different with other analytes.

Single nanoporous gold nanowire was introduced as a tunable one-dimensional nano-sensor platform with both SERS and catalytic activity, and it precisely fit the requirement of materials for in situ SERS monitoring of plasmon-assisted catalytic reaction. The nanoporous gold nanowires exhibited much more "hot spots" on their surface and much better SPR effect than the smooth nanowires. We demonstrated that these nanowires could be used as a SERS substrate assuring the sensitivity and reproducibility of Raman signals. Besides, they could be applied as a kind of heterogeneous catalyst for in situ SERS monitoring of the plasmon-assisted catalytic reaction-reduction of p-nitrothiophenol (p-NTP) to p,p-dimercaptoazobenzene (DMAB) at their surface. The SERS and catalytic activity of the nanowires could be respectively optimized by adjusting their dealloying time, similar to the procedure of catalyst screening.

Ethanol to butanol (ETB) process is an attractive route to upgrade abundant substrate into a product with superior properties in energy industry. For the mechanism research, the detection of intermediates is vital. In this paper, intermediates in ETB reaction catalyzed by monoxide ions [ZrO]+/[CeO]+/[TiO]+ were investigated by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). Some important organic small molecule intermediates, metal-adduct intermediates and their formation pathways were discussed to reveal the reaction mechanism in this research. Isotopic labelling experiments were also designed to ensure the existence of these intermediates.

We have combined droplet extraction and a pulsed direct current electrospray ionization mass spectrometry method (Pico-ESI-MS) to obtain information-rich metabolite profiling from single cells. We studied normal human astrocyte cells and glioblastoma cancer cells. Over 600 tandem mass spectra (MS2) of metabolites from a single cell were recorded, allowing the successful identification of more than 300 phospholipids. We found the ratios of unsaturated phosphatidylcholines (PCs) to saturated PCs were significantly higher in glioblastoma cells compared to normal cells. In addition, both isomeric PC (17:1) and (phosphatidylethanolamine) PE (20:1) were found in glioblastoma cells, whereas only PC (17:1) was observed in astrocyte cells. Our method paves the way to characterize the chemical contents of single cells, providing rich metabolome information. We suggest that this technique is general and can be applied to other life science studies such as differentiation and drug resistance of individual cells.

Gold nanoparticles (AuNPs)-embedded paper was coupled with ion-transmission mass spectrometry (MS) to enable the highly sensitive detection of chemical warfare agent (CWA) simulants in solutions. With the assistance of a low-temperature plasma (LTP) probe, we found that AuNPs were capable to enhance the ionization efficiencies of target analytes, with MS signal intensities surprisingly undergone an 800-fold increase under optimized conditions. The interaction between AuNPs and the radiofrequency electromagnetic field was believed to promote the desorption/ionization process, resulting in the unusual signal enhancement phenomenon. Based on this finding, we established a method for the rapid analysis of two simulants of nerve agents, dimethyl methylphosphonate (DMMP) and diisopropyl methylphosphonate (DIMP), with a dynamic range from 0.5 ng/mL to 100 ng/mL and detection limits of 0.1 ng/mL and 0.3 ng/mL, respectively. As sample pretreatments have been eliminated, the developed strategy is particularly promising for the on-site detection of CWAs considering its simple and rapid analytical workflow.

Inductively Coupled Plasma Mass Spectrometry (ICP-MS) based immunoassay method has been proposed in multiple immunoassays but has not been used in competitive and sandwich formats immunoassay simultaneously. The two immunoassays were usually conducted separately in clinical field depending on the size and the amount of binding sites of targets. We proposed an immunoassay method based on magnetic beads and ICP-MS detection that could be suitable for both small and large molecules. The functionalized magnetic beads were added to capture the immune complex after immune reaction. In this way, thyrotropin and free thyroid hormone can be captured, separated and then detected according to the elemental tags by ICP-MS simultaneously. The new method was evaluated by comparing the results with time resolved fluorescence immunoassays (TRFIA). The dynamic ranges of TSH and FT4 were 0.16-105 mU/L and 3.5-65 pmol/L, respectively. The limits of detections were 0.06 mU/L for TSH and 1.59 pmol/L for FT4. And the relative standard deviations (RSD) of TSH and FT4 were 4.64% at 2.5 mU/L and 1.87% at 5.85 pmol/L. This immunoassay method enables the determination of small and large biomolecules simultaneously via competitive and sandwich immunoassay formats.

The synthesis, properties and applications of a water-soluble boronate-functioned hemicyanine–naphthol hybrid as a novel ratiometric fluorescent sensor for hydrogen peroxide are presented. The dye displayed remarkable a colour change from pale orange (λem = 590 nm) to pink (λem = 690 nm) in the presence of H2O2, which could be rationalized by the chemoselective H2O2-mediated transformation of arylboronate to phenolate with high selectivity and a fast response (within 2 min). A good linear relationship (R2 = 0.9951) was obtained with the H2O2 concentration ranging from 0 to 25 μM, with a limit of detection of 0.09 μM according to the signal-to-noise ratio (S/N = 3). The advantages of this fluorophore include easy modification, excellent aqueous solubility and superior photostability, and it has been applied to the detection of trace amounts of hydrogen peroxide in water samples. Copyright © 2015 John Wiley & Sons, Ltd.

A new atmospheric pressure ionization method termed pyroelectricity-assisted infrared laser desorption ionization (PAI-LDI) was developed in this study. The pyroelectric material served as both sample target plate and enhancing ionization substrate, and an IR laser with wavelength of 1064 nm was employed to realize direct desorption and ionization of the analytes. The mass spectra of various compounds obtained on pyroelectric material were compared with those of other substrates. For the five standard substances tested in this work, LiNbO3 substrate produced the highest ion yield and the signal intensity was about 10 times higher than that when copper was used as substrate. For 1-adamantylamine, as low as 20 pg (132.2 fmol) was successfully detected. The active ingredient in (Compound Paracetamol and 1-Adamantylamine Hydrochloride Capsules), 1-adamantylamine, can be sensitively detected at an amount as low as 150 pg, when the medicine stock solution was diluted with urine. Monosaccharide and oligosaccharides in Allium Cepa L. juice was also successfully identified with PAI-LDI. The method did not require matrix-assisted external high voltage or other extra facility-assisted set-ups for desorption/ionization. This study suggested exciting application prospect of pyroelectric materials in matrix- and electricity-free atmospheric pressure mass spectrometry research.

Methylmercury (CH3Hg) is one of the most widespread toxic contaminants. Although the formation of CH3Hg in aqueous environments has been widely investigated, little information is available on direct anthropogenic or natural emissions of CH3Hg to the atmosphere. In this work, ICP-MS/MS was chosen as a tool to study abiotic methylation of inorganic mercury reacting with VOCs in a gas environment for the first time. We found that the gaseous Hg+ ions were transformed to the more toxic CH3Hg+ ions instantaneously when colliding with some VOCs. Several VOCs, e.g., methyl iodide (CH3I), methylbenzene, acetic acid and ethyl acetate, exhibited good methylation of Hg+ ions with productivities of 1.77%, 1.28%, 1.35% and 1.18%, respectively. Four isotope peaks of CH3199Hg (M = 214), CH3200Hg (M = 215), CH3201Hg (M = 216) and CH3202Hg (M = 217) were identified when Hg+ ions collided with CH3I, and the methyl group in CH3Hg+ was validated by the source of CD3I, indicating that CH3Hg+ ions were formed. This study reveals that the abiotic methylation of Hg+ ions could occur when in contact with the VOCs in the atmosphere, leading to secondary environmental pollution.

A new “turn-on” fluorescent probe, composed of a protected phenol group with a p-nitrobenzyl moiety that functions as a latent donor and conjugated with two benzo[f]indolinium acceptors, was developed and applied for imaging nitroreductase (NTR) in hypoxic tumor cells. Nitrobenzyl moieties on the substrate can be conveniently converted into aminobenzyl groups with a nitroreductase-catalyzed reaction in the presence of reduced nicotinamide adenine dinucleotide (NADH). This is followed by a cleavage reaction and the release of the free phenol moiety, which is manifested in enhanced fluorescence intensity during the detection process. Our experimental results show that the NTR detection ability is over 100 equivalents of other biological reductants (1 mM), whereas the confocal fluorescence imaging of tumor cells indicates the possibility of its application in biomedical research fields for tumor hypoxia diagnosis.

Herein we report a novel strategy for the detection and identification of proteins using unmodified noble metal nanoparticles. Five gold nanoparticles (AuNPs) and two silver nanoparticles (AgNPs) with different sizes were utilized as sensing elements to create a colorimetric sensor array. In the presence of proteins, the UV-vis absorbance of the noble metal nanoparticles changed due to the interactions between the protein and nanoparticles, producing distinct absorbance response patterns which can be visually detected by the naked eye. The color pattern of the array is a unique "fingerprints" for each protein sample, which can be differentiated by linear discriminant analysis (LDA). Ten different proteins at concentrations of 0.5, 5 and 50 μM have be successfully discriminated. Moreover, the array was also able to discriminate different bacteria at a concentration of 0.05 OD in 200 μL, as well as cancer cells at the level of 5000 cells in 200 μL. This work demonstrates that an unmodified noble metal nanoparticle-based protein detection array has potential for applications in medical diagnostics.

Unsaturated fatty acids (FAs) serve as nutrients, energy sources, and signaling molecules for organisms, which are the major components for a large variety of lipids. However, structural characterization and quantitation of unsaturated FAs by mass spectrometry remain an analytical challenge. Here, we report the coupling of epoxidation reaction of the C═C in unsaturated FAs and tandem mass spectrometry (MS) for rapid and accurate identification and quantitation of C═C isomers of FAs in a shotgun lipidomics approach. Epoxidation of the C═C leads to the production of an epoxide which, upon collision induced dissociation (CID), produces abundant diagnostic ions indicative of the C═C location. The total intensity of the same set of diagnostic ions for one specific FA C═C isomer was also used for its relative and absolute quantitation. The simple experimental setup, rapid reaction kinetics (2 min), high reaction yield (90% for monounsaturated FAs), and easy-to-interpret tandem MS spectra enable a promising methodology particularly for the analysis of unsaturated FAs in complex biological samples such as human plasma and animal tissues.

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) has been used in imaging of small molecules (500 Da) in fingerprints, such as gunshot residues and illicit drugs. However, identifying and mapping relatively high mass molecules are quite difficult owing to insufficient ion yield of their molecular ions. In this report, graphene oxide (GO)-enhanced TOF-SIMS was used to detect and image relatively high mass molecules such as poison, alkaloids (600 Da) and controlled drugs, and antibiotics (700 Da) in fingerprints. Detail features of fingerprints such as the number and distribution of sweat pores in a ridge and even the delicate morphology of one pore were clearly revealed in SIMS images of relatively high mass molecules. The detail features combining with identified chemical composition were sufficient to establish a human identity and link the suspect to a crime scene. The wide detectable mass range and high spatial resolution make GO-enhanced TOF-SIMS a promising tool in accurate and fast analysis of fingerprints, especially in fragmental fingerprint analysis.

Plasmon-enhanced fluorescence (PEF) generally requires the samples settled on a metal substrate and the effective enhancement distance is less than 100 nm, which limit its application in intracellular sensing. Herein, we report a nano endoscopy with PEF effect for sensing analytes inside the extremely small volume samples. The nano endoscopy was fabricated by assembling single nanoporous gold nanowire (PGNW) on the tip of a tungsten needle. It was accurately manipulated to insert into a micro droplet, and an effective sensing was realized at micrometre scale with submicrometer resolution. By taking lysozyme as a model sensing target, a 23-fold improvement of sensitivity was obtained, comparing with that of smooth gold nanowire (SGNW). These results indicated that the nano endoscopy can realize a high spatial resolution sensing, showing its potential application in intracellular sensing.

Low-abundance metabolites or proteins in single-cell samples are usually undetectable by mass spectrometry (MS) due to the limited amount of substances in single cells. This limitation inspired us to further enhance the sensitivity of commercial mass spectrometers. Herein, we developed a technique named repeated ion accumulation by ion trap MS, which is capable of enhancing the sensitivity by selectively and repeatedly accumulating ions in a linear ion trap for up to 25 cycles. The increase in MS sensitivity was positively correlated with the number of repeated cycles. When ions were repeatedly accumulated for 25 cycles, the sensitivity of adenosine triphosphate detection was increased by 22-fold within 1.8 s. Our technique could stably detect low-abundance ions, especially MSn ions, at the single-cell level, such as 5-methylcytosine hydrolyzed from sample equivalent to ∼0.2 MCF7 cell. The strategy presented in this study offers the possibility to aid single-cell analysis by enhancing MS detection sensiti...