RESUMEN
Mass spectrometry (MS) is a powerful tool for exploring single-cell heterogeneity. However, due to the ultralow absolute content of most substances in a single cell, existing methods can only analyze high-content substances; conventional methods are incompetent for quantitative analysis of important trace-amount small-molecule metabolites such as ammonia and sulfide. Herein, a method integrating single-cell extraction, online derivatization, and MS for multifunctional and more accurate MS analysis is reported. For application, ammonia content in a single cell was analyzed, where the cellular heterogeneity in ammonia metabolism was revealed. First, the extraction room of a microfluidic probe was covered on the target single cell, and the extraction fluid was allowed to flow through a single cell and dissolve cellular ammonia. Then, the ammonia was mixed and reacted with the pretreatment reagent input from another inlet to achieve the derivatization and signal amplification, enhancing the analysis sensitivity on MS. Finally, the sample was sent to MS, and the ammonia content was successfully quantitatively evaluated by analyzing its derivative urotropine, demonstrating the potential of this method to advance single-cell mass spectrometry analysis to higher sensitivity.
Asunto(s)
Amoníaco , Microfluídica , Cromatografía de Gases y Espectrometría de Masas/métodos , Espectrometría de Masas en Tándem/métodos , Indicadores y ReactivosRESUMEN
Precise sampling of undissolved chemical components from subcellular regions of living single cells is a prerequisite for their in-depth analysis, which could promote understanding of subtle early stage physiological or pathological processes. Here we report a microfluidic method to extract undissolved components from subcellular regions for MS analysis. The target single cell was isolated by the microchamber beneath the microfluidic probe and washed by the injected biocompatible isotonic glucose aqueous solution (IGAS). Then, the sampling solvent was injected to extract undissolved components from the expected subcellular region of the living single cell, where the position and size of the sampling region could be controlled. The components immobilized by undissolved cellular structures were proven to be successfully extracted. Since unextracted subcellular regions were protected by IGAS, the single cell could survive after a tiny part was extracted, providing the possibility of repetitive sampling of the same living cell. Phospholipids extracted from the subcellular regions were successfully identified. The results demonstrated the feasibility of our method for subcellular sampling and analysis.
Asunto(s)
Microfluídica , Fosfolípidos , Microfluídica/métodos , Espectrometría de Masas , Análisis de la Célula Individual/métodosRESUMEN
Microglia are the main immune cells in the brain playing a critical role in neuroinflammation, and numerous pieces of evidence have proved that energy metabolism is closely associated with inflammation in activated microglia. Salidroside (Sal) isolated from Tibetan medicine Rhodiola crenulate can inhibit microglial hypoxia inflammation (HI). However, whether the inhibition is due to the intervening energy metabolic process in microglia is not clear. In this work, the hypoxic microenvironment of BV2 microglial cells was simulated using deferoxamine (DFO) in vitro and the change of cell metabolites (lactate, succinate, malate, and fumarate) was real-time online investigated based on a cell microfluidic chip-mass spectrometry (CM-MS) system. Meanwhile, for confirming the metabolic mechanism of BV2 cells under hypoxia, the level of HI-related factors (LDH, ROS, HIF-1α, NF-κB p65, TNF-α, IL-1ß, and IL-6) was detected by molecular biotechnology. Integration of the detected results revealed that DFO-induced BV2 cell HI was associated with the process of energy metabolism, in which cell energy metabolism changed from oxidative phosphorylation to glycolysis. Furthermore, administration of Sal treatment could effectively invert this change, and two metabolites of Sal were identified: tyrosol and 4-hydroxyphenylacetic acid. In general, we illustrated a new mechanism of Sal for reducing BV2 cell HI injury and presented a novel analysis strategy that opened a way for real-time online monitoring of the energy metabolic mechanism of the effect of drugs on cells and further provided a superior strategy to screen natural drug candidates for HI-related brain disease treatment.
Asunto(s)
Microfluídica , Microglía , Glucósidos , Humanos , Hipoxia , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Espectrometría de Masas , Microglía/metabolismo , FN-kappa B/metabolismo , Fenoles , Transducción de SeñalRESUMEN
Multicellular tumor spheroids (MCTSs) are in vitro solid tumor models with physiological relevance. To achieve robust process control, a MCTS fabrication method that combines cell membrane engineering and droplet microfluidic techniques is designed. The fluidic control and the chemical interactions between biotin and streptavidin enable artificial cell aggregation to be accomplished in seconds. Then, spheroids with a uniform size are fabricated within alginate microcapsules. Microfluidic mixing-based cell aggregation regulates the cell aggregate size and the spheroid composition, and the microcapsules regulate the size of spheroids from 120 to 180 µm. The method shows applicability for various cancer cell lines, including HCT116, HepG2, and A549. In addition, composite colon cancer spheroids consisting of HCT116 and NIH3T3 with predetermined cell ratios and uniform distributions are produced. The generated MCTSs are assessed using the ELISA and UPLC-MS/MS techniques. The release of vascular endothelial growth factor (VEGF) and the 5-fluorouracil (5-FU) resistance differ in the monotypic and cocultured colon cancer models. Our method provides a robust way to produce consistent and customized MCTSs in cancer research and drug screening.
Asunto(s)
Neoplasias del Colon , Factor A de Crecimiento Endotelial Vascular , Animales , Ratones , Humanos , Cápsulas , Cromatografía Liquida , Células 3T3 NIH , Espectrometría de Masas en Tándem , Fluorouracilo/farmacología , Esferoides Celulares , Dispositivos Laboratorio en un Chip , Línea Celular TumoralRESUMEN
Confinement of chemical species in a controllable micrometer-level (several to a dozen micrometers) space in an aqueous environment is essential for precisely manipulating chemical events in subcellular regions. However, rapid diffusion and hard-to-control micrometer-level fluids make it a tough challenge. Here, a versatile open microfluidic method based on an aqueous two-phase system (ATPS) is developed to restrict species inside an open space with micron-level width. Unequal standard chemical potentials of the chemical species in two phases and space-time correspondence in the microfluidic system prevent outward diffusion across the phase interface, retaining the target species inside its preferred phase flow and creating a sharp boundary with a dramatic concentration change. Then, the chemical flow (the preferred phase with target chemical species) is precisely manipulated by a microfluidic probe, which can be compressed to a micron-level width and aimed at an arbitrary position of the sample. As a demonstration of the feasibility and versatility of the strategy, chemical flow is successfully applied to subcellular regions of various kinds of living single cells. Subcellular regions are successfully labeled (cytomembrane and mitochondria) and damaged. Healing-regeneration behaviors of living single cells are triggered by subcellular damage and analyzed. The method is relatively general regarding the species of chemicals and biosamples, which could promote deeper cell research.
RESUMEN
A highly enantio- and diastereoselective [4 + 1]-annulation reaction between α,ß-unsaturated imines and allylic carbonates has been realized under the catalysis of a novel hybrid P-chiral phosphine oxide-phosphine, providing enantioenriched polysubstituted 2-pyrrolines in good to excellent yields and up to 99% ee. Based on Han's methods, the catalyst featuring a sole P(O)-chirality in the molecule is readily accessible and represents a class of new chiral phosphine organocatalysts. In the plausible catalytic mechanism, an intramolecular Coulombic interaction between the in situ generated phosphonium cation and polar chiral PâO moiety may play a positive role.