Intact living-cell electrolaunching ionization mass spectrometry for single-cell metabolomics.

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.

Complement-Opsonized NIR-IIb Emissive Immunotracers for Dynamically Monitoring Neutrophils in Inflammation-Related Diseases.

Real-time monitoring of neutrophil dynamics is crucial for timely diagnosis and effective treatment of inflammation-related diseases, which requires a reliable tracer for in vivo tracking of neutrophils. However, immunotracers for neutrophils are extremely limited because of the difficulty in labeling the cells. Inspired by the natural biological function of the complement system, a strategy of enhancing the complement C3 opsonization of lanthanide-doped nanoparticles (LnNPs) by modulating their surface chemistry, thus developing a near infrared-IIb emissive nanotracer for neutrophils, is reported herein. Four kinds of surface-modified LnNPs are fabricated, among which phospholipids DOPG-modified LnNPs (LnNPs@PG) with weak antifouling ability and hydroxyl groups adsorb more complement C3 proteins and form covalent linkages with C3b active fragments under inflammation conditions, inducing enhanced complement C3 opsonization. Therefore, LnNPs@PG with enhanced complement C3 opsonization are capable of efficiently labeling inflammation-stimulated neutrophils in vivo through complement-receptors-mediated phagocytosis and achieve dynamic monitoring neutrophils during cutaneous wound healing and cerebral ischemia/reperfusion.

Analytical methods for obtaining binding parameters of drug-protein interactions: A review.

The study of drug-protein interactions can reveal the corresponding binding mechanisms, providing valuable information for the early phase drug development and development of new drugs. This article reviews the methods used for obtaining the binding parameters of drug-protein systems. The methods include equilibrium dialysis, high-performance affinity chromatography, capillary electrophoresis, spectroscopy, calorimetry, competition and displacement, mass spectrometry, fluorescence resonance energy transfer, and thermal stability shift analysis. Relevant parameters include the association constant, number of binding sites, thermodynamic properties, binding force types, binding site types, binding distances, changes in protein conformation, and changes in protein stability. In addition, the review also summarizes the principles, advantages, and limitations of each method in detail. The comparison of parameter information can not only guide method selection but also provide valuable reference information for in-depth exploration of drug-protein interaction mechanisms.

Controllable Fabrication of Small-Size Holding Pipets for the Nondestructive Manipulation of Suspended Living Single Cells.

The capture and manipulation of single cells are an important premise and basis for intracellular delivery, which provides abundant molecular and omics information for biomedical development. However, for intracellular delivery of cargos into/from small-size suspended living single cells, the capture methods are limited by the lack of small-size holding pipets, poor cell activity, and the low spatial accuracy of intracellular delivery. To solve these problems, a method for the controllable fabrication of small-size holding pipets was proposed. A simple, homemade microforge instrument including an imaging device was built to cut and melt the glass capillary tip by controlling the heat production of a nichrome wire. The controllable fabrication of small-size holding pipets was realized by observing the fabrication process in real time. Combined with an electroosmotic drive system and a micromanipulation system with high spatial resolution, the holding pipet achieved the active capture, movement, and sampling of suspended living single cells. Moreover, solid-phase microextraction was performed on captured single pheochromocytoma cells, and the extracted dopamine was successfully detected using an electrochemical method. The homemade microforge instrument overcame the limitations of traditional microforges, resulting in holding pipets that were sufficiently small for small-size suspended single living cells (5-30 µm). This proactive capture method overcame the shortcomings of existing methods to achieve the multiangle, high-precision manipulation of single cells, thereby allowing the intracellular delivery of small-size single cells in suspension with high spatiotemporal resolution.

Visually precise, low-damage, single-cell spatial manipulation with single-pixel resolution.

The analysis of single living cells, including intracellular delivery and extraction, is essential for monitoring their dynamic biochemical processes and exploring intracellular heterogeneity. However, owing to the 2D view in bright-field microscopy and optical distortions caused by the cell shape and the variation in the refractive index both inside and around the cells, achieving spatially undistorted imaging for high-precision manipulation within a cell is challenging. Here, an accurate and visual system is developed for single-cell spatial manipulation by correcting the aberration for simultaneous bright-field triple-view imaging. Stereo information from the triple view enables higher spatial resolution that facilitates the precise manipulation of single cells. In the bright field, we resolved the spatial locations of subcellular structures of a single cell suspended in a medium and measured the random spatial rotation angle of the cell with a precision of ±5°. Furthermore, we demonstrated the visual manipulation of a probe to an arbitrary spatial point of a cell with an accuracy of <1 pixel. This novel system is more accurate and less destructive for subcellular content extraction and drug delivery.

Solid-phase microextraction integrated nanobiosensors for the serial detection of cytoplasmic dopamine in a single living cell.

Dopamine participates in many physiological and pathological processes. Dynamic monitoring of dopamine levels in the cytoplasm of a single living cell reflects not only the functional state of dopamine synthesis factors but also the processes of related neurodegenerative diseases. Due to the low content of cytoplasmic dopamine and the difficulty to keep cells alive during the operating process, the detection of cytoplasmic dopamine is still challenging. Herein, a solid-phase microextraction (SPME) technique integrated nanobiosensor was employed to trace and quantify dopamine concentration fluctuations in the cytoplasm of a single living cell. We designed a polypyrrole modified carbon fiber nanoprobe as a bifunctional nanoprobe that can extract cytoplasmic dopamine and then perform electrochemical detection. This bifunctional nanoprobe can detect 10 pmol/L extracted dopamine and detected a 60% decrease of the cytoplasmic dopamine concentration in a single living cell by K+ stimulation. This study allowed for the first time serially detecting cytoplasmic dopamine while keeping the target cell alive, which might yield a new method for research on dopamine neurotoxicity and the related drug action mechanisms for neurodegenerative disease.

Controllable fabrication of pico/femtoliter pipette sampling probes and visual sample volume determination.

We propose a hydraulically assisted eddy-current etching method for the controllable fabrication of pico/femtoliter sampling probes with equal inner diameters along the length of the probe. The relative standard deviations of the outer and inner diameters (O.D. and I.D., respectively) of several 1.07-µm-I.D. sampling probe tips fabricated in a single batch using this method were 2.2% and 2.8%, respectively, and the average O.D./I.D. ratio of these probes was 1.17. The probe fabrication method has high reproducibility, and sharp tips are produced, which is advantageous for the transfer of ultra-small sample volumes. Further, the narrow, equal diameter, cylindrical inner bore allows the online, visual determination of the pipetted sample volume by utilizing a microscopic imaging system to measure the liquid length. This value is converted linearly to the pipetted volume, whose measurement error is on a sub-pixel level. Image-based sampling using the fabricated probes was achieved by connecting the probe to an electroosmotic pump, which allowed the controlled pipetting of pico/femtoliter samples. Because the pipette allows samples of the same volume to be measured accurately, the pipette was applied for the semiquantitative mass spectrometry analysis of the metabolites in individual epidermal cells sampled from different parts of Portulaca oleracea plants. The results show that different types of cells have distinct metabolite profiles. Further, the experiments showed that, in dopamine-rich leaves, the content of dopamine in the petioles is higher than that in the foliage. The probe fabrication strategy opens new avenues for the controllable pipetting of ultra-small volume samples and the realization of the visual pipetting of pico/femtoliter samples.

Gas phase reaction between chromones and solvent in an electrospray ionization source.

Chromones were measured by using electrospray ionization mass spectrometry in negative mode. Interestingly, in addition to the deprotonated ion ([M - H]- ), unexpected [M + 17]- and [M + 31]- ions were observed in high intensity when water and methanol were used as the solvent. Chromones with different substitutes were tested. Compared with the deprotonated ion, [M + 17]- and [M + 31]- ions were observed with higher abundances when the C-3 site of chromones was substituted by electron withdrawing groups. Based on high performance liquid chromatography-mass spectrometry (LC-MS), deuterium-labeling and collisional-induced dissociation experiments, a covalent gas-phase nucleophilic addition reaction between chromone and water, and the formation of a noncovalent complex between chromone and methanol were proposed as the mechanism for the observed [M + 17]- and [M + 31]- ions, respectively. Understanding and using these unique gas phase reactions can avoid misannotation when analyzing chromones and their metabolites.

Loss of benzaldehyde in the fragmentation of protonated benzoylamines: Benzoyl cation as a hydride acceptor in the gas phase.

In electrospray ionization tandem mass spectrometry of protonated 1-benzoylamines (1-benzoylpiperadine, 1-benzoylmorpholine, and 1-benzoyl-4-methylpiperazine), the dominant fragmentation pathway was amide bond cleavage to form benzoyl cation and neutral amine. Meanwhile, in their fragmentations, an interesting loss of benzaldehyde (106 Da) was observed and identified to derive from hydride transfer reaction between the benzoyl cation and amine. A stepwise mechanism for loss of 106 Da (benzene and CO) could be excluded with the aid of deuterium labeling experiment. Theoretical calculations indicated that hydride transfers from amines (piperadine, morpholine, and 1-methylpiperazine) to benzoyl cation were thermodynamically permitted, and 1-methylpiperazine was the best hydride donor among the 3 amines. The mass spectrometric experimental results were consistent with the computational results. The relative abundance of the iminium cation (relative to the benzoyl cation) in the fragmentation of protonated 1-benzoyl-4-methylpiperazine was higher than that in the fragmentation of the other 2 protonated 1-benzoylamines. By comparing the fragmentations of protonated 1-benzyl-4-methylpiperazine and protonated 1-benzoyl-4-methylpiperazine and the energetics of their hydride transfer reactions, this study revealed that benzoyl cation was a hydride acceptor in the gas phase, but which was weaker than benzyl cation.

Pico-HPLC system integrating an equal inner diameter femtopipette into a 900 nm I.D. porous layer open tubular column.

A picoflow high performance liquid chromatography (pico-HPLC) system was developed, which could directly pipette femtoliter samples using a separation column tip driven by an electroosmotic pump. Amino acid enantiomers were separated in the 900 nm I.D. porous layer open tubular column at a flow rate of 13.50 pL min-1.