High-Throughput Metabolite Analysis of Unicellular Microalgae by Orthogonal Hybrid Ionization Label-Free Mass Cytometry.

Microalgae metabolite analysis is fundamental for the rational design of metabolic engineering strategies for the biosynthesis of high-value products. Mass spectrometry (MS) has been utilized for single-cell microalgae analysis. However, limitations in the detection throughput and polarities of detectable substances make it difficult to realize high-throughput screening of high-performance microalgae. Herein, a plasma-assisted label-free mass cytometry, named as PACyESI-MS, was proposed combining the advantages of orthogonal hybrid ionization and high-throughput MS analysis, which realized rapid metabolite detection of single microalgae. The cell detection throughput of PACyESI-MS was up to 52 cells/min. Dozens of the critical primary and secondary metabolites within single microalgae were detected simultaneously, including pigments, lipids, and energy metabolites. Furthermore, metabolite changes of Chlamydomonas reinhardtii and Haematococcus pluvialis under nitrogen deficiency stress were studied. Discrimination of Chlamydomonas under different nutrient conditions was realized using single-cell metabolite profiles obtained by PACyESI-MS. The relationships between the accumulation of bioactive astaxanthin and changes in functional primary metabolites of Haematococcus were investigated. It was demonstrated that PACyESI-MS can detect the flexible change of metabolites in single microalgae cells under different nutritional conditions and during the synthesis of high-value products, which is expected to become an important tool for the design of metabolic engineering-based high-performance microalgae factories.

Single-Cell Unsaturated Lipid Profiling for Studying Chemoresistance Heterogeneity of Triple-Negative Breast Cancer Cells.

Chemoresistance to triple-negative breast cancer (TNBC) is a critical issue in clinical practice. Lipid metabolism takes a unique role in breast cancer cells; especially, unsaturated lipids involving cell membrane fluidity and peroxidation are highly remarked. At present, for the lack of a high-resolution molecular recognition platform at the single-cell level, it is still hard to systematically study chemoresistance heterogeneity based on lipid unsaturation proportion. By designing a single-cell mass spectrometry workflow based on CyESI-MS, we profiled the unsaturated lipids of TNBC cells to evaluate lipidomic remodeling under platinum stress. Profiling revealed the heterogeneity of the polyunsaturated lipid proportion of TNBC cells under cisplatin treatment. A cluster of cells identified by polyunsaturated lipid accumulation was found to be involved in platinum sensitivity. Furthermore, we found that the chemoresistance of TNBC cells could be regulated by fatty acid supplementation, which determinates the composition of unsaturated lipids. These discoveries provide insights for monitoring and controlling cellular unsaturated lipid proportions to overcome chemoresistance in breast cancer.

Microfluidic Impedance Cytometry Enabled One-Step Sample Preparation for Efficient Single-Cell Mass Spectrometry.

Single-cell mass spectrometry (MS) is significant in biochemical analysis and holds great potential in biomedical applications. Efficient sample preparation like sorting (i.e., separating target cells from the mixed population) and desalting (i.e., moving the cells off non-volatile salt solution) is urgently required in single-cell MS. However, traditional sample preparation methods suffer from complicated operation with various apparatus, or insufficient performance. Herein, a one-step sample preparation strategy by leveraging label-free impedance flow cytometry (IFC) based microfluidics is proposed. Specifically, the IFC framework to characterize and sort single-cells is adopted. Simultaneously with sorting, the target cell is transferred from the local high-salinity buffer to the MS-compatible solution. In this way, one-step sorting and desalting are achieved and the collected cells can be directly fed for MS analysis. A high sorting efficiency (>99%), cancer cell purity (≈87%), and desalting efficiency (>99%), and the whole workflow of impedance-based separation and MS analysis of normal cells (MCF-10A) and cancer cells (MDA-MB-468) are verified. As a standalone sample preparation module, the microfluidic chip is compatible with a variety of MS analysis methods, and envisioned to provide a new paradigm in efficient MS sample preparation, and further in multi-modal (i.e., electrical and metabolic) characterization of single-cells.

Covalent Organic Framework Nanosheets for Fluorescence Quantification of Peptide.

Rapid and sensitive quantification of peptides plays an important role in clinical diagnosis. Fluorescence assay is one of the most promising peptide detection tools, but it relies on intrinsic fluorescence or additional derivatization, resulting in poor versatility. Covalent organic frameworks (COFs) have shown a good application prospect in the field of fluorescence detection, but their application scope is limited to heavy metal ions and some small polar organic molecules. Herein, we report the application of COFs nanosheet for fluorescence detection of peptides. Fluorescent sp2 acrylonitrile-linked COFs nanosheets (TTAN-CON) were prepared by water-assisted ultrasonic exfoliation which performed with excellent fluorescence properties with Stokes shifts of 146 nm and fluorescence quantum yield of up to 24.45%. Compared to the bulk fluorescent COFs, exfoliated CONs films performed with better stability of fluorescence signal in solution. We found the fluorescence of TTAN-CON can be effectively quenched by hydrophobic peptides at a very rapid rate (less than 5 min per sample). TTAN-CON presented good sensitivity and selectivity for hydrophobic peptides detection via the static and dynamic joint quenching mechanism. TTAN-CON was further used to detect NLLGLIEAK and ProGRP31-98, two target peptide fragments of lung cancer biomarker ProGRP. The fluorescence intensities of TTAN-CON were negative linearly correlated with the amounts of hydrophobic NLLGLIEAK over the range of 5-1000 ng/mL with the correlation coefficients over 0.99, and the limit of detection was 1.67 ng/mL, displaying higher sensitivity and convenience than traditional optical methods. What's more, the quantification of ProGRP31-98 was achieved by the quantification of hydrophobic peptides in its enzyme hydrolysis products. We anticipate COFs nanosheets to be a universal fluorescence detection work-box for peptides biomarkers with clinical significance.

A Rapid SARS-CoV-2 Nucleocapsid Protein Profiling Assay with High Sensitivity Comparable to Nucleic Acid Detection.

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-1. It is 1-2 orders of magnitude lower than existing commercial antigen ELISA kits, which is comparative to or superior than the PCR based nucleic acid testing. Owing to its rapidity, ultrasensitivity, as well as easy operation, it holds great promise as a tool for screening of COVID-19 and other epidemics such as monkey pox.

Determination of testosterone in serum by magnetic molecularly imprinted polymer-coupled nano-ESI-MS.

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.

The simultaneous quantitative detection of multiple hormones based on PS-MS: affinity capture by a single antibody.

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-D3 (T-D3) as an internal standard. Standard curves for standard solution or spiked serum samples were realized in the range of 0.01-2 µg L-1. The LODs for the three androgens were 2.3 × 10-3 µg L-1 for testosterone, 4.6 × 10-3 µg L-1 for androstenedione, and 2.8 × 10-3 µg L-1 for androsterone. The recovery results verified the reliability and stability of our method. This strategy has widespread potential for advancing the combination of immunoassay and MS methods in the analysis of small molecules, with high through-put and low cost.

The heterogeneity of oxidized lipids in individual tumor cells reveals NK cell-mediated cytotoxicity by label-free mass cytometry.

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.

Site-Specific Scissors Based on Myeloperoxidase for Phosphorothioate DNA.

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.

Rapid Disulfide Mapping in Peptides and Proteins by meta-Chloroperoxybenzoic Acid (mCPBA) Oxidation and Tandem Mass Spectrometry.

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.

Discriminating Leukemia Cellular Heterogeneity and Screening Metabolite Biomarker Candidates using Label-Free Mass Cytometry.

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.

Combination of Structured Illumination Microscopy with Hyperspectral Imaging for Cell Analysis.

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.

Uncover Single Nanoparticle Dynamics on Live Cell Membrane with Data-Driven Historical Experience Analysis.

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.

Dynamic Monitoring of Phase-Separated Biomolecular Condensates by Photoluminescence Lifetime Imaging.

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.

In Situ Identification and Spatial Mapping of Microplastic Standards in Paramecia by Secondary-Ion Mass Spectrometry Imaging.

Microplastics (MPs) are universally present in the ecosystem and pose great threats to the environment and living organisms. Research studies have shown that small MPs (<50 µm in diameter) are especially toxic and account for more than half of all MPs collected in the Atlantic Ocean. Nevertheless, current methods for the detection and analysis of MPs are incapable of achieving rapid and in situ analysis of small MPs in the biota to ultimately enable the study of their biological effects. In this work, we report a method that allows rapid in situ identification and spatial mapping of small MPs directly from paramecia with high accuracy by acquiring chemical composition information using secondary-ion mass spectrometry (SIMS) imaging. Specifically, six types of common MPs (polymethyl methacrylate, polyvinyl chloride, polypropylene, polyethylene terephthalate, polyglycidyl methacrylate, and polyamide 6) with a diameter of 1-50 µm were simultaneously imaged with high chemical specificity at a spatial resolution of 700 nm. In situ spatial mapping of a group of MPs ingested by paramecia was performed using SIMS fragments specific to the plastic composition with no sample pretreatment, revealing the aggregation of MPs in paramecia after ingestion. Compared with existing methods, one additional advantage of the developed method is that the MPs and the organism can be analyzed in the same experimental workflow to record their fingerprint spectra, acquiring biochemical information to evaluate MP fate, toxicity, and the MP-biota interaction.

Simultaneous multicolour imaging using quantum dot structured illumination microscopy.

Multicolour structured illumination microscopy (SIM) is a powerful tool used for the investigation of the dynamic interaction between subcellular structures. Nevertheless, most of the multicolour SIM schemes are currently limited by conventional fluorescent dyes and wavelength-dependent optical systems, and can only sequentially record images of different colour channels instead of obtaining multicolour datasets simultaneously. To address these issues, we present a novel multicolour SIM scheme referred to as quantum dot structured illumination microscopy (QD-SIM). QD-SIM enables simultaneously excitation and collection of multicolour fluorescent signals. We also propose a theoretical analysis of the image formation in two-dimensional multicolour SIM to help combine the optically sectioned and super-resolution attributes of SIM. Based on this theory, QD-SIM enables optically sectioned, super-resolution, multicolour simultaneous imaging at a single plane.

A multiplex bacterial assay using an element-labeled strategy for 16S rRNA detection.

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.

Metabolic fingerprinting of cell types in mouse skeletal muscle by combining TOF-SIMS with immunofluorescence staining.

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.

Thickness-dependent Young's modulus of polycrystalline α-PbO nanosheets.

Litharge, in two dimensional (2D) nanostructure form, has recently ignited considerable theoretical interest due to its excellent photoelectric and magnetic properties. However, the lack of an efficient synthesis method hinders its development. Here, we provide an interfacial solvothermal strategy for controllably synthesizing ultrathin hexagonal polycrystalline α-PbO nanosheets in micrometer scale. This strategy can also be utilized for the synthesis of other 2D materials. Experimental atomic force microscope nanoindentation measurements reveal the relationship between the thickness of polycrystalline α-PbO nanosheets and the corresponding Young's modulus, expressed as E = E0 + Kt -1. First-principles calculation supports the result and ascribes the cause to interlayer sliding from particular weak interlayer interactions. Additionally, the enhanced mechanical strength of the polycrystalline structure compared to its single-crystal counterpart is attributed to the alternate arrangement of grain-boundaries effects. The summative formula may be extended to other 2D materials with weak interlayer interactions, which has the potential to provide guidance for constructing flexible devices.

Development and evaluation of an element-tagged immunoassay coupled with inductively coupled plasma mass spectrometry detection: can we apply the new assay in the clinical laboratory?

Introduction Element-tagged immunoassay coupled with inductively coupled plasma-mass spectrometry (ICP-MS) detection has the potential to revolutionize immunoassay analysis in clinical detection; however, a systematic evaluation with the standard guidelines of the assay is needed to ensure its performance meets the requirements of the clinical laboratory. Methods Carcinoembryonic antigen (CEA) was chosen for analysis using the proposed method. A systematic evaluation of the proposed assay was carried out according to the Clinical and Laboratory Standards Institute (CLSI). The 469 clinical samples were analyzed using the new method and compared with the electrochemiluminescent immunoassay (ECLIA) method. Results The measurement range of the assay was 1-900 ng/mL, with a detection limit of 0.83 ng/mL. The inter-assay and intra-assay imprecision were 4.67% and 5.38% with high concentration samples, and 9.27% and 17.64% with low concentration samples, respectively. The cross-reactivity (%) for different antigens was less than 0.05%, and the recovery was between 94% and 108%. Percentage deviation of all the dilutions was less than 12.5% during linearity estimation. The interference bias caused by different substances was less than 10%. The reference interval of the assay was 0-4.442 ng/mL. Comparison with the commercial ECLIA method for clinical sample detection, the proposed method showed a correlation of 0.9878 and no significant differences between the methods were observed (p = 0.6666). Conclusions The ICP-MS based immunoassay was successfully developed, and the analytical performance of the assay met the requirements of the CLSI, which fully proved the clinical transferability and application of the new method.