In Situ Capturing and Counting Device for the Specific Depletion and Purification of Cancer-Derived Exosomes

From metabolic waste to biological mediators, exosomes have emerged as the key player in a variety of pathological processes, particularly in oncogenesis. The exosome-mediated communication network involves nearly every step of cancer progression, promoting the proliferation and immune escape of cancer cells. Therefore, the removal of cancer-derived exosomes has profound clinical significance. Current methods for exosome separation and enrichment are either for large-scale samples or require complex pretreatment processes, lacking effective methods for trace-volume exosome capture in situ. Herein, we have developed an in situ exosome capturing and counting device based on the antibody-functionalized capillary. Specific antibodies targeting exosome biomarkers were immobilized to the inner wall of the capillary via biotin-streptavidin interaction for direct cancer exosome capturing. Subsequent exosome staining enabled imaging and enumeration. Acceptable linearity and reproducibility were achieved with our device, with the capturing and detective range between 3.3 × 104 and 3.3 × 108 particles, surpassing the nanoparticle tracking analysis by 2 orders of magnitude while requiring merely 30 μL sample. We demonstrated that MCF-7-derived exosomes induced epithelial–mesenchymal transition of epithelial cells MCF-10A, and our method was able to completely or partially reverse the transition by complete depletion or specific depletion of cancer exosomes without any preprocessing. Moreover, both whole exosomes and cancer-specific exosomes alone from mimic blood samples were successfully captured and counted, without obvious non-specific adsorption. In all, our approach realized the in situ depletion and number-counting of cancer-derived exosomes directly from the complex humoral environment, having the potential to provide a comprehensive tumor therapeutic and prognosis evaluation tool by targeted hemodialysis and counting of tumor-derived exosomes

Novel Nitrocellulose Membrane Substrate for Efficient Analysis of Circulating Tumor Cells Coupled with Surface-Enhanced Raman Scattering Imaging

The capture and detection of circulating tumor cells (CTCs) in the bloodstream of patients with cancer is crucial for the clinical diagnosis and therapy. In the present work, a facile and integrated approach based on novel nitrocellulose membrane substrate and large-scale surface-enhanced Raman scattering (SERS) imaging technology has been developed for CTCs’ sensitive detection and enumeration. The system mainly consists of three aspects: capture of CTCs in bloodstream, SERS probes labeling of the captured CTCs and large-scale SERS imaging readout of CTCs enumeration. The NC membrane was used to prepare the novel CTC-capture substrate through antibody self-assembled. It was low-cost, easily prepared and completely nontoxic. Furthermore, excellent capture efficiency of the substrate was demonstrated using nonsmall-cell lung cancer (NSCLC) cells (NCI-H1650) as target cells. As the most sensitive detection technology, SERS holds huge potential in CTCs analysis. Large-scale SERS imaging was employed in CTCs enumeration for the first time, instead of the conventional fluorescence imaging. Our SERS probes, with a simplified structure, offered highly enough sensitivity to recognize every single cell clearly. In the simulation experiment of spiking 100 cancer cells into 1 mL of human whole blood, 34 cells were captured and counted successfully according to the SERS imaging result. Our experimental results demonstrate the potential feasibility of novel NC membrane substrate coupled with large-scale SERS imaging technology for the accurate enumeration of CTCs in human whole blood

Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis

Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future

Array-Based Online Two Dimensional Liquid Chromatography System Applied to Effective Depletion of High-Abundance Proteins in Human Plasma

In this work, an array-based online two-dimensional liquid chromatography (2D-LC) system was constructed for protein separation and effective depletion of high-abundance proteins in human plasma. This system employed a strong anion exchange column in the first dimension and eight reversed-phase liquid chromatographic columns in the second dimension. All the protein components in the first dimension were enriched on the trapping columns, simultaneously back-flushed and concurrently separated in the second dimension. LC eluents were then collected on 96-well plates for further analysis. Compared with common 2D-LC system, this system showed an 8-fold increase in throughput and convenient utilization of stop-flow mode for sample separation. The RSD of retention time and peak area were separately below 0.51% and 8%. Recovery rates of four standard proteins were all above 95%. This array-based 2D-LC system was subsequently applied to the analysis of proteins in human plasma. The eluents containing high-abundance proteins were rapidly located according to the results of bicinchoninic acid assay. In all, with the effective depletion of 84 high-abundance proteins, a total of 1332 proteins were identified through our system. The dynamic range of the identified protein concentrations covered 9 orders of magnitude, ranging from 41 g/L level for HSA down to 0.01 ng/mL level for the low-abundance proteins

Magnetic Binary Metal–Organic Framework As a Novel Affinity Probe for Highly Selective Capture of Endogenous Phosphopeptides

Highly efficient detection of endogenous phosphopeptides from complex biosamples is essential in phosphopeptidomics analysis due to the severe disturbance caused by the chaotic biological environment. In this study, for highly selective capture of endogenous phosphopeptides, a magnetic binary metal–organic framework (MOF) with Zr–O and Ti–O centers (denoted as Fe₃O₄@PDA@Zr-Ti-MOF) was designed and synthesized by a facile postsynthetic method. Briefly, Zr-based MOF was first coated on the surface of magnetic Fe₃O₄ with polydopamine (PDA) as a linker, and then, the as-prepared Fe₃O₄@PDA@Zr-MOF was exposed to DMF solution containing TiCl₄(THF)₂, resulting in the successful synthesis of Fe₃O₄@PDA@Zr-Ti-MOF. This newly prepared Fe₃O₄@PDA@Zr-Ti-MOF owned the merits of large specific surface area, unique porous structure, and superparamagnetism as well as the enhanced dual affinities of Zr–O and Ti–O centers toward both endogenous mono-phospho-peptides and multi-phospho-peptides, showing highly improved performance with better selectivity and sensitivity compared to single-metal centered MOFs (Fe₃O₄@PDA@Zr-MOF, Fe₃O₄@PDA@Ti-MOF). The Fe₃O₄@PDA@Zr-Ti-MOF was also successfully applied to extract endogenous phosphopeptides in biological sample of human saliva. As a result, 34 mono-phosphorylated peptides and 10 multi-phosphorylated peptides were detected from merely 1 μL of pristine human saliva, confirming its bright prospects in phosphopeptidomics analysis

Quantification of GPC1(+) Exosomes Based on MALDI-TOF MS In Situ Signal Amplification for Pancreatic Cancer Discrimination and Evaluation

Pancreatic cancer (PC) has a high mortality, with a fairly low five-year survival rate, because of its delayed diagnosis. Recently, liquid biopsy, especially based on exosomes, has attracted vast attention, thanks to its low invasiveness. Herein, we constructed a protocol for pancreatic cancer related Glypican 1 (GPC1) exosome quantification, based on in situ mass spectrometry signal amplification, by utilizing mass tag molecules on gold nanoparticles (AuNPs). Exosomes were extracted and purified by size-exclusion chromatography (SEC), captured by TiO2 modified magnetic nanoparticles, and then targeted specifically by anti-GPC1 antibody modified on AuNPs. With matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), the signal of PC biomarker, GPC1, was converted to a mass tag signal and amplified. With addition of a certain amount of internal standard molecules modified on AuNPs, the relative intensity ratio of mass tag to internal standard was proportional to the concentration of GPC1(+) exosomes derived from pancreatic cancer cell lines, PANC-1, with good linearity (R2 = 0.9945) in a wide dynamic range from 7.1 × 10 to 7.1 × 106 particles/μL. This method was further applied to plasma samples from healthy control (HC) and pancreatic cancer patients with different tumor load, and exhibited a great potential in discriminating diagnosed PC patients from HC, and has the monitoring potential in PC progression

New Method for Counting and Picking Out Single Circulating Tumor Cells from Microliter-Volume Samples for Tumor Progression Surveillance and Single-Cell Heterogeneity Analysis

Circulating tumor cells (CTCs) are crucial in tumor progression and metastasis, but the knowledge of their roles grows slowly at single-cell levels. Characterizing the rarity and fragility of CTCs by nature, highly stable and efficient single-CTC sampling methods are still lacking, which impedes the development of single-CTC analysis. Herein, an improved, capillary-based single-cell sampling (SiCS) method, the so-called bubble-glue single-cell sampling (bubble-glue SiCS), is introduced. Benefiting from the characteristic that the cells tend to adhere to air bubbles in the solution, single cells can be sampled with bubbles as low as 20 pL with a self-designed microbubble-volume-controlled system. Benefiting from the excellent maneuverability, single CTCs are sampled directly from 10 μL volume of real blood samples after fluorescent labeling. Meanwhile, over 90% of the CTCs obtained survived and well proliferated after the bubble-glue SiCS process, which showed considerable superiority for downstream single-CTC profiling. Furthermore, a highly metastatic breast cancer model of the 4T1 cell line in vivo was employed for the real blood sample analysis. Increases in CTC numbers were observed during the tumor progression process, and significant heterogeneities among individual CTCs were discovered. In all, we propose a novel avenue for target SiCS and provide an alternative technique route for CTC separation and analysis

Metathesis Reaction-Induced Significant Improvement in Hydrogen Storage Properties of the KF-Added Mg(NH₂)₂–2LiH System

The hydrogen storage properties and mechanisms of the Mg­(NH₂)₂–2LiH system with potassium halides (KF, KCl, KBr, and KI) were investigated and discussed. The results show that the KF-added sample exhibits superior hydrogen storage properties as ∼5.0 wt % of hydrogen can be reversibly stored in the 0.08KF-added sample via a two-stage reaction with an onset dehydrogenation temperature of 80 °C. However, hydrogen storage behaviors of the samples with KCl, KBr, and KI remain almost unchanged. The fact that KF can readily react with LiH to convert to KH and LiF due to the favorable thermodynamics during ball milling should be the primary reason for its significant effects, as the presence of KH provides a synergetic thermodynamic and kinetic destabilization in the hydrogen storage reaction of the Mg­(NH₂)₂–2LiH system by declining the activation energy of the first-step dehydrogenation as a catalyst and reducing the desorption enthalpy change of the second step as a reactant. The understanding on the role played by KF sheds light on how to further decrease the operating temperature and enhance the hydrogen storage kinetics of the metal–N–H system

Novel Strategy of High-Abundance Protein Depletion Using Multidimensional Liquid Chromatography

In this study, for the first time, a comprehensive two-dimensional (2D) liquid-phase separation system, coupling strong cation exchange chromatography (SCX) to reversed-phase high performance liquid chromatography (RPLC), instead of specificity depletion method, was developed at the intact protein level for depletion of high-abundance proteins from rat liver. Proteins were prefractionated by SCX in the first dimensional separation, followed by RPLC with high resolution separation. UV absorption intensity was used to differentiate high-abundance proteins. The proteins with the absorbance intensity above 0.1 AU were defined as high abundance proteins and depleted. After removal of high-abundance proteins; other proteins were pooled, digested, and subsequently separated by capillary liquid chromatography coupled with MALDI-TOF/TOF mass spectrometry analysis. The high efficiency of the strategy was demonstrated by analyzing the soluble protein extracted from rat liver tissue. In total, 77 high-abundance proteins were depleted in one experiment flow. The ratio of depleted content of high-abundance proteins to that of total proteins was about 34.5%. In total, 1530 proteins were identified using the depletion strategy. Quantitative estimation of high-abundance proteins through liquid chromatography combined with UV absorption spectra was achieved. On the basis of the reproducible experimental results, a rapid and high-throughput depletion protocol was put forward. Along with depletion of the most (79.1%) high-abundance proteins and the separation of digested peptides, the total separation time could be less than 30 h. This strategy has no bias for depleting high-abundance proteins and enhances the number of identified proteins; therefore, it can be widely used in the global proteins analysis. Keywords: strong cation exchange chromatography • reversed-phase high performance liquid chromatography • proteome • high-abundance proteins • middle- and low-abundance protein