Facile Preparation of Three-Dimensional Wafer with Interconnected Porous Structure for High-Performance Capture and Nondestructive Release of Circulating Tumor Cells.

Efficient isolation and downstream bioinformation analysis of circulating tumor cells (CTCs) in whole blood contribute to the early diagnosis of cancer and investigation of cancer metastasis. However, the separation and release of CTCs remain a great challenge due to the extreme rarity of CTCs and severe interference from other cells in complex clinical samples. Herein, we developed a low-cost and easy-to-fabricate aptamer-functionalized wafer with a three-dimensional (3D) interconnected porous structure by grafting polydopamine (PDA), poly(ethylene glycol) (PEG), and aptamer in sequence (Ni@PDA-PEG-Apt) for the capture and release of CTCs. The Ni@PDA-PEG-Apt wafer integrated the features of Ni foam with a 3D interconnected porous structure offering enough tunnels for cells to flow through and enhancing aptamer-cell contact frequency, the spacer PEG with flexible and high hydrophilic property increasing anti-interference ability and providing the wafer with more binding sites for aptamer, which result in an enhanced capture specificity and efficiency for CTCs. Because of these advantages, the Ni@PDA-PEG-Apt wafer achieved a high capture efficiency of 78.25%. The captured cancer cells were mildly released by endonuclease with up to 61.85% efficiency and good proliferation. Furthermore, tumor cells were injected into mice and experienced circulation in vivo. In blood samples after circulation, 65% of target tumor cells can be efficiently captured by the wafer, followed by released and recultured cells with high viability. Further downstream metabolomics analysis showed that target cancer cells remained with high biological activity and can be well separated from MCF-10A cells based on metabolic profiles by the PCA analysis, indicating the great potential of our strategy for further research on the progression of cancer metastasis. Notably, not only is the wafer cheap with a cost of only 3.58 U.S. dollars and easily prepared by environmental-friendly reagents but also the process of capturing and releasing tumor cells can be completed within an hour, which is beneficial for large-scale clinical use in the future.

Facile preparation of a novel chitosan-derived porous graphitized carbon biomaterial for highly efficient capture of N-glycans.

The comprehensive characterization of N-glycans is of significant importance for the discovery of potential biomarkers and the diagnosis and therapy of diseases. Herein, we designed and fabricated a porous graphitized carbon biomaterial (CS-900-1C) using cheap and available chitosan as the carbon source via a facile pyrolysis process and a post-oxidation strategy for the effective capture of N-glycans. Thanks to its large surface area (2846 m2 g-1), high graphitization degree, suitable oxidation degree and unique porous structure, the CS-900-1C biomaterial exhibits an ultralow detection limit (1 ng µL-1), an excellent size-exclusion effect (OVA digest : BSA protein : OVA protein, 1 : 1000 : 1000, w/w/w) and satisfactory reusability (at least 8 cycles) in the capture of standard N-glycans. Moreover, CS-900-1C has successfully been applied in profiling the difference of N-glycans during diabetes progression (obesity, impaired glucose tolerance, diabetes patients and healthy control) where we discovered that the expression levels of five N-glycans show a gradually increasing trend as diabetes progresses. Remarkably, the five specific N-glycans could be considered as biomarkers to accurately diagnose the progression of diabetes. Our work not only developed a novel porous graphitized carbon biomaterial for the large-scale characterization of N-glycans but also provided new guidance for the precise therapy of diabetes.

Titanium(IV)-functionalized zirconium-organic frameworks as dual-metal affinity probe for recognition of endogenous phosphopeptides prior to mass spectrometric quantification.

A zirconium-organic framework was modified with titanium(IV) ions to obtain a modified framework that is shown to be a viable sorbent for selective capture of phosphopeptides. This dual-metal affinity probe exhibits 0.1 fM limits of detection and excellent size-exclusion effect (the mass ratio of ß-casein digests/BSA/intact ß-casein is 1:1000:1000). This is attributed to abundant Ti(IV) and Zr(IV) coordination sites and high porosity. The performance of the sorbent for extracting endogenous phosphopeptides from human serum and saliva was investigated. Especially, 105 endogenous phosphopeptides from saliva were captured specifically. In addition, the amino acid frequency of the enriched phosphopeptides was analyzed. Conservation of sequence around the identified phosphorylated sites from saliva confirmed that phosphorylation took place in the proline-directed motifs. Graphical abstractSchematic representation of a method for the specific enrichment of phosphopeptides by a modified metal-organic framework. Following size-exclusion elution, the phosphopeptides are quantified by mass spectrometry.

Surface-enhanced Raman scattering (SERS) imaging-guided real-time photothermal ablation of target cancer cells using polydopamine-encapsulated gold nanorods as multifunctional agents.

In this study, we developed a novel "see-and-treat" theranostic system named "surface-enhanced Raman scattering (SERS) imaging-guided real-time photothermal therapy" for accurate cancer detection and real-time cancer cell ablation using the same Raman laser. Facilely synthesized polydopamine-encapsulated gold nanorods (AuNRs), which possess excellent biocompatibility and enhanced stability, were used as multifunctional agents. Under near-infrared (NIR) laser irradiation, polydopamine-encapsulated AuNRs show strong SERS effect and high photothermal conversion efficiency simultaneously. After immobilization of antibodies (anti-EpCAM), polydopamine-encapsulated gold nanorods show high specificity to target cancer cells. Tumor margins could be distinguished facilely by a quick SERS imaging process, which was confirmed by H&E staining results. By focusing the exciting light on detected cancer cells for a prolonged time, cancer cells could be ablated immediately without the need of other procedure. This "see-and-treat" theranostic strategy combining SERS imaging and real-time photothermal therapy using the same Raman laser is proposed for the first time. Experimental results confirmed the feasibility of our "SERS imaging-guided real-time photothermal therapy system." This novel theranostic strategy can significantly improve the efficiency of cancer therapy in clinical application, allowing the effective ablation of cancer cells with no effects on surrounding healthy tissues. Graphical abstract ᅟ.

Synthesis of bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets for sequential selective enrichment of phosphopeptides and glycopeptides for mass spectrometric analysis.

In this work, the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets were designed and synthesized for the sequential selective enrichment of phosphopeptides and glycopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the nanosheets were successfully applied to the enrichment of phosphopeptides and glycopeptides sequentially, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine ß-casein diluted to 0.02 ng/µL (8 × 10(-16) mol/µL) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 0.1 ng/µL (2.5 × 10(-15) mol/µL). The enrichment selectivity of the bifunctional nanosheets was evaluated by capturing phosphopeptides from a peptide mixture of ß-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (8.3 × 10(-12) mol of ß-casein and 8.3 × 10(-9) mol of BSA in 100 µL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:50 (5.0 × 10(-11) mol of HRP and 2.5 × 10(-9) mol of BSA in 100 µL). Graphical Abstract A workflow of the sequential enrichment strategy for phosphopeptides and glycopeptides by the bifunctional TiO2@SiO2-B(OH)2@Fe3O4@TiO2 sandwich-like nanosheets.

Membrane protein isolation and identification by covalent binding for proteome research.

A novel method to achieve highly efficient identification of membrane proteins (MPs) has been developed based on a covalent binding (CB) strategy. For this purpose, magnetic nanoparticles coated with a PEG layer were synthesized. The PEG chain end was functionalized to form the PEG-tresyl group, which is an octopus-like long arm to capture the free amino groups of MPs. The long arm could be used to bind proteins in a high concentration of the SDS medium. Then, the SDS and interfering substances were completely depleted by washing. The CB proteins could form a molecular monolayer on the surface of the nanoparticles in the denatured state, which was significantly favorable for the proteolysis of MPs. Therefore, isolation with CB and highly efficient digestion resulted in a larger scale of MPs. The method has been verified by a proteome identification of mouse liver samples. A total of 2946 MPs were identified in an MP fraction. A total of 1505 proteins were characterized as integral MPs, and 735 MPs were identified beyond the largest database summarized by PeptideAtlas. This approach has great potential for membrane proteome research.

Magnetic capture of polydopamine-encapsulated Hela cells for the analysis of cell surface proteins.

A novel method to characterize cell surface proteins and complexes has been developed. Polydopamine (PDA)-encapsulated Hela cells were prepared for plasma membrane proteome research. Since the PDA protection, the encapsulated cells could be maintained for more than two weeks. Amino groups functionalized magnetic nanoparticles were also used for cell capture by the reaction with the PDA coatings. Plasma membrane fragments were isolated and enriched with assistance of an external magnetic field after disruption of the coated cells by ultrasonic treatment. Plasma membrane proteins (PMPs) and complexes were well preserved on the fragments and identified by shot-gun proteomic analytical strategy. 385 PMPs and 1411 non-PMPs were identified using the method. 85.2% of these PMPs were lipid-raft associated proteins. Ingenuity Pathway Analysis was employed for bio-information extraction from the identified proteins. It was found that 653 non-PMPs had interactions with 140 PMPs. Among them, epidermal growth factor receptor and its complexes, and a series of important pathways including STAT3 pathway were observed. All these results demonstrated that the new approach is of great importance in applying to the research of physiological function and mechanism of the plasma membrane proteins. SIGNIFICANCE: This work developed a novel strategy for the proteomic analysis of cell surface proteins. According to the results, 73.3% of total identified proteins were lipid-raft associated proteins, which imply that the proposed method is of great potential in the identification of lipid-raft associated proteins. In addition, a series of protein-protein interactions and pathways related to Hela cells were pointed out. All these results demonstrated that our proposed approach is of great importance and could well be applied to the physiological function and mechanism research of plasma membrane proteins.

Selective enrichment of glycopeptides/phosphopeptides using Fe3O4@Au-B(OH)2@mTiO2 core-shell microspheres.

In this work, the bifunctional Fe3O4@Au-B(OH)2@mTiO2 core-shell core-shell microspheres were designed and synthesized for the selective enrichment of glycopeptides/ phosphopeptides. Due to the bifunctional property of the titanium dioxide and the boronic acid group, the microspheres were successfully applied to the enrichment of phosphopeptides and glycopeptides, evaluated by capturing phosphopeptides from tryptic digestion of model phosphoprotein bovine ß-casein diluted to 2.0pgµL-1 (8.0×10-17molµL-1) and glycopeptides from tryptic digestion of model glycoprotein horseradish peroxidase (HRP) diluted to 80pgµL-1 (2.0×10-15molµL-1). The enrichment selectivity of the bifunctional microspheres was evaluated by capturing phosphopeptides from a peptide mixture of ß-casein and bovine serum albumin (BSA) with the molar ratio of 1:1000 (4.2×10-12mol of ß-casein and 4.2×10-9mol of BSA in 100µL) and glycopeptides from a peptide mixture of HRP and BSA up to the ratio of 1:100 (5.0×10-12mol of HRP and 5.0×10-10mol of BSA in 100µL).

Facile synthesis of thiol and alkynyl contained SERS reporter molecular and its usage in assembly of polydopamine protected bioorthogonal SERS tag for live cell imaging.

A bioorthogonal Raman reporter-embedded Au-core and polydopamine-shell nanoprobe was initially designed and synthesized for live cell surface-enhanced Raman scattering (SERS) imaging in Raman-silent region. The firstly synthetic bioorthogonal Raman reporter (E)-2- ((4-(phenylethynyl) benzylidene)amino)ethanethiol (PBAT) provided intense Raman signal at 2220cm(-1) in Raman-silent region. And its synthetic method was environmentally benign, high yield and easy in operation. In addition, the hydrophobicity of PBAT led to slightly aggregation of gold nanoparticles, which enhance the SERS intensity of the nanoprobe through hot spots. Furthermore, owing to the remarkable biocompatibility of polydopamine (PDA), the SERS nanoprobe can be smoothly internalized into cancer cells to realize SERS imaging in Raman-silent region. Finally, the SERS mapping results confirmed that nanoprobe exhibited strong SERS signal intensity, excellent stability in complex biological environment and low toxicity inside live cancer cells. Therefore, the reporter-embedded core-shell nanoprobe has enormous potential of applications in biomedical diagnostics in the near future.

An effective and in-situ method based tresyl-functionalized porous polymer material for enrichment and digestion of membrane proteins and its application in extraction tips.

Membrane proteins are one of promising targets for drug discovery because of the unique properties in physiological processes. Due to their low abundance and extremely hydrophobic nature, the analysis of membrane proteins is still a great challenge. In this work, an effective and in-situ method were developed to enrich and digest membrane proteins by adopting tresyl-functionalized porous polymer material. With tresyl groups, the material can effectively immobilize membrane proteins via covalent bonding on the surface. The material became a facile carrier to enrich membrane proteins from the rat liver in detergents and organic solvents owing to its outstanding binding capacity and excellent biocompatibility. Moreover, it was further applied in extraction tips to capture and in-situ digest the pretreatment membrane proteins in two different solutions. A total of 600 membrane proteins (51% of total protein groups) and 359 transmembrane proteins were identified by nano-LC-ESI-MS/MS in 4% sodium dodecyl sulfate (SDS), and similar results were achieved in the 60% methanol solution. All these results demonstrated that the new approach is of great promise for large-scale characterization of membrane proteins.

Multilayer Hydrophilic Poly(phenol-formaldehyde resin)-Coated Magnetic Graphene for Boronic Acid Immobilization as a Novel Matrix for Glycoproteome Analysis.

Capturing glycopeptides selectively and efficiently from mixed biological samples has always been critical for comprehensive and in-depth glycoproteomics analysis, but the lack of materials with superior capture capacity and high specificity still makes it a challenge. In this work, we introduce a way first to synthesize a novel boronic-acid-functionalized magnetic graphene@phenolic-formaldehyde resin multilayer composites via a facile process. The as-prepared composites gathered excellent characters of large specific surface area and strong magnetic responsiveness of magnetic graphene, biocompatibility of resin, and enhanced affinity properties of boronic acid. Furthermore, the functional graphene composites were shown to have low detection limit (1 fmol) and good selectivity, even when the background nonglycopeptides has a concentration 100 fold higher. Additionally, enrichment efficiency of the composites was still retained after being used repeatedly (at least three times). Better yet, the practical applicability of this approach was evaluated by the enrichment of human serum with a low sample volume of 1 µL. All the results have illustrated that the magG@PF@APB has a great potential in glycoproteome analysis of complex biological samples.

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.

[A novel and facile microchip based on nitrocellulose membrane toward efficient capture of circulating tumor cells].

A novel and facile circulating tumor cell (CTC) microchip has been developed for the isolation and detection of cancer cells. The CTC microchip was prepared based on the nitrocellulose membrane substrate, which shows high affinity to proteins and hence can adsorb antibodies naturally. We employed non-small-cells of lung cancer NCI-H1650 as target cells and testified the high capture efficacy of the CTC microchip. Furthermore, we spiked 500 cancer cells to 1 mL healthy donor's whole blood in order to simulate the detection of CTC in patient and detected 182 cancer cells ultimately, indicating the huge application potential in the future.