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.

•The adding of Raman reporter twice contributed to enhancing the Raman intensity.•Numerous Ag nanoparticles self-assembly were arranged around gold core surface.•Core-shell satellites structure showed 10-fold increase in the Raman intensity.A novel, high–sensitivity, biocompatible SERS tag with core-shell structure based on gold nanoparticles containing alkynyl molecule core -silver nanoparticle satellites shell was fabricated for the first time to be used for live cancer cells Surface enhanced Raman scattering (SERS) imaging. (E)-2-((4-(phenylethynyl)benzylidene) amino) ethanethiol (PBAT) synthesized facilely in our lab is the Raman-silence region reporter which is advantage for bioorthogonal SERS cell imaging. In order to enhance the intensity of the Raman tags for live cancer cell imaging, a series of news measures have been adopted. Firstly, reporter molecules of the PBAT were added twice, which is embedded in the gold core with the reduction of tetrachloroaurate and then PBAT is conjugated again on disperse gold nanoparticles (PBAT-Au). Furthermore, numerous Ag nanoparticles self-assembly were densely arranged around PBAT-Au core surface (PBAT- Au@Ag), just like a circle of satellites cluster, which produce obvious “hot spots” effects enhancing the signal of the Raman tags enormously. Finally, Bovine serum albumin (BSA) and polydopamine (PDA) coated on the PBAT- Au@Ag successively, defined as (PBAT-Au@Ag@BSA@PDA), which make as-synthesized nanocomposites own features of bio-compatibility and facilitates antibody modification. Compared with Au@PBAT@PDA, PBAT-Au@Ag@BSA@PDA with core-shell satellites structure showed 10-fold increase in the Raman signals intensity. Moreover, PBAT-Au@Ag@BSA@PDA nanocomposites were successfully applied in the Raman imaging of human glioma cells (U251) by the recognition of the anti-epidermal growth factor receptor (EGFR). All experimental results demonstrated that the nanocomposites have high value and huge potential application in the live cancer cells imaging and biomedical diagnostics in the near future.A new SERS tag with core-shell structure based on gold nanoparticles core containing alkynyl molecule and silver nanoparticle satellites shell, was designed and synthesized for live cancer cells Surface enhanced Raman scattering (SERS) imaging in Raman-silence region. The tag shows 10-fold increase in the Raman signals intensity.Download high-res image (184KB)Download full-size image

The development of additional functions and applications of covalent organic framework (COF)-derived materials still remains highly desired. In our work, a novel COF-functionalized magnetic graphene biocomposite (MagG@COF-5) was first developed as an ultrasensitive hydrophilic matrix via a facile self-assembly method for efficiently recognizing N-linked glycopeptides. By integrating the characteristics of the magnetic graphene and COF-5 layer, the MagG@COF-5 owns features of an outstanding magnetic response, a high specific area, strong hydrophilic properties and a unique size-exclusion effect. Accordingly, the MagG@COF-5 biocomposite showed excellent performance in N-linked glycopeptide analysis with a low detection limit (0.5 fmol μL−1), an excellent size-exclusion effect (HRP digests/BSA, 1 : 600), good recyclability and reusability. More excitingly, the practical applicability of the biocomposite was evaluated by treatment with human serum (1 μL), in which 232 N-linked glycopeptides from 85 glycoproteins were detected. All the results demonstrate that the as-synthesized MagG@COF-5 biocomposite has huge potential for use in glycoproteome and clinical diagnosis fields. It will also open up new phases for application of COF-based materials.

Protein N-glycosylation is a ubiquitous and important post-translational modification that has been involved in the development and progression of a series of human-related diseases. Until recently, the highly selective capturing of glycopeptides from complex biosamples was still significant and challenging work due to their changeable structures, ultralow abundance, and strong ion-suppressing effect. Here we first report the preparation and characterization of a novel, hydrophilic, porous biocomposite composed of magnetic graphene functionalized with metal–organic frameworks (MOFs) (MG@Zn-MOFs) able to recognize glycopeptides. Thanks to its strong magnetic responsiveness, large specific surface area, excellent biocompatibility, and unique size-exclusion effect, the MG@Zn-MOFs showed outstanding sensitivity and selectivity and good recyclability in glycopeptides analysis. More excitingly, in practical application, 517 N-glycopeptides within 151 unique glycoproteins were clearly identified from human serum (1 μL) treated with the MG@Zn-MOFs, which is the best result among published reports so far. All the results demonstrate the promising commercialized usage of the biocomposite for the enrichment of glycopeptides in complex samples through a convenient and efficient process. Furthermore, it is anticipated that our strategy may offer promising guidance to develop new biocomposites functionalized with bio-MOFs for glycoproteomic applications.Keywords: enrichment; glycoproteomics; human serum; hydrophilic pore; mass spectrometry; metal−organic frameworks (MOFs)-functionalized biocomposite

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.

•The novel hydrophilic polyamidoxime polymers were successfully synthesized.•The materials had efficiency for sequential enrichment of glycol- and phosphopeptide.•The performance of the materials was tested by standard peptides and human serum.Selective enrichment of glycopeptides or phosphopeptides with great biological significance is essential for high-throughput mass spectrometry analysis. However, most previously reported methods only focused on enriching either glycopeptides or phosphopeptides rather than enriching them both. In this work, for the first time, a facile route was developed for the synthesis of polyamidoxime polymers with intrinsic hydrophilic skeletons and attractive long chain structure. The polyamidoxime materials (co-PAN) were synthesized from polyacrylonitrile (PAN) precursor and were successfully used for selective enrichment of glycopeptides. After that, co-PAN as a matrix functionalized with titanium ions (co-PAN@Ti4+) could efficiently enrich phosphopeptides. The performances of the polymers for sequential selective and effective enrichment of glycopeptides and phosphopeptides were evaluated with standard peptide mixtures and human serum. Moreover, the efficiency of enrichment of the material was still retained after being used repeatedly. These results demonstrated that the polymers showed great potential in the practical application of proteomics.

•A novel ZIFs-functionalized magnetic carbon-graphene material was synthesized.•The material exhibits excellent the ability to trap N-linked glycans of biosamples.•48 N-linked glycans from human serum treated with the material were identified.•All results demonstrate that the material has good performance for N-linked glycans.The highly selective enrichment of N-linked glycans from complex biological sample is still very important but challenging task due to the ultra-low abundance, complicated structures and strong ion suppress effect caused by distractors such as proteins, peptides and salts. Here, we firstly present a novel metal–organic frameworks (MOFs)-functionalized magnetic nanoporous carbon-graphene composites (C-magG@ZIF-8) synthesized through a smart process. The obtained materials enjoy the unique properties including strong magnetic responsiveness, a large sum of graphitized carbon pore, remarkable biocompatibility and large specific surface area. By virtue of these unique properties, the C-magG@ZIF-8 composites displayed excellent selectivity and sensitivity, good recyclability and incredible size exclusion ability (roughly 2000 times) in the N-linked glycans analysis. Furthermore, 48 N-linked glycans were clearly identified from the normal human serum treated with the C-magG@ZIF-8. There is reason to believe that our smart strategy offers new possibilities for preparing the MOFs-functionalized composites for large-scale characterization of glycoproteomics by mass spectrometry analysis.

•A pollen-based honeycomb MOAC material with atomic-level-doped binary metal oxides was synthesized.•The hydrophilic material achieved excellent performance in enrichment of phosphopeptides.•The material has considerable potential for large-scale characterization of phosphoproteomics.The enrichment and separation of phosphopeptides from mixed biological samples is a technologically very significance, but highly challenging work. Current designed materials are mainly based on the broad and effective adsorptive character of metal oxide affinity chromatography (MOAC). Though significant progress has been made in the enrichment of phosphopeptides with MOAC material, there are chances for further development. In this study, a novel pollen-based MOAC honeycomb material was firstly explored in which the suitable hydrophilic channels preferentially enrich much more endogenous phosphopeptides than nonphosphopeptides or proteins while doping binary metal oxides at the atomic level and the ultra-high specific surface area have further allowed it to possess more effective active sites. Based on these unique features, the pollen-based material exhibited high selectivity for β-casein (mass ratio of β-casein/BSA, 1:1500), ultra-low detection limit (0.1 fmol), desirable reusability. Moreover, the bionics MOAC composites were investigated in the enrichment of phosphopeptides from nonfat milk, human serum (male and female at the same age) and mice liver, results of which indicate the great potential of the composite for the phosphoproteome analysis of complex biological samples through the cheap and environmentally friendly process.

•The 3D network structure can trap cells and increase contact probability.•The usage of dendrimers has assisted in improving the capture efficiency.•The substrate which is soft and ultra-hydrophilic can keep the viability of cells.Highly efficient isolation of living tumor cells possesses great significance in research of cancer. Hence, we have designed the 3-aminophenylboronic acid (APBA) derivative dendrimer-functionalized 3D network polyacrylamide/poly (methyl methacrylate) copolymer as capture substrate which is easily prepared, template free and low-cost. The structure of copolymer is compared to “fishing net” in order to increase the contact between cells and substrates. The application of poly (amidoamine) dendrimers provides abundant amino groups to react with APBA which is just like “baits” that can bond with sialic acid in the cytomembrane to realize cell capture. The 3D network structure trammels cancer cells, offers great reaction space and displays hydrophilic surface, which has immensely improved the contact probability of cells and materials. Due to the 3D network structure and dendrimer, this material can achieve a high capture efficiency of 87±5% in 45 min. The viability of captured cells is nearly 100%, as a result of the soft and hydrophilic surface and hypotoxicity of this copolymer.In this work, we have successfully designed and synthesized the dendrimers modified ultra-hydrophilic copolymer with the trapping network structure and plenty of reaction sites to enhance the affinity of materials and target cells, the synthesized materials can achieve highly efficiency of cell capture, meanwhile keep the cell ability.

•One-step synthetic method of thiol and alkynyl contained bioorthogonal SERS reporter is environmentally benign, high yield and easy in operation.•Au@PBAT@PDA nanoparticles exhibit strong SERS signal intensity, excellent stability in complex biological environment.•Au@PBAT@PDA nanoparticles show good imaging results for live cancer cell SERS imaging in Raman-silent region.•The rich functional groups (i.e., catechol and amine) on PDA surface provide a broad platform for further modification in biomedical application.•A Raman-silent region nanoprobe was synthesized for live cell SERS 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 2220 cm−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.

In recent MS-based glycoproteomics, the selective enrichment of glycopeptides from complex biological samples is essential. In this work, for the first time, a novel ultra hydrophilic dendrimer-modified magnetic graphene@polydopamine@poly(amidoamine) (magG@PDA@PAMAM) was synthesized via three simple and rapid steps. The magnetic composites combined large surface of graphene, strong magnetic responsiveness of Fe3O4 with double hydrophilic abilities of PDA and PAMAM. Especially PAMAM had long dendritic chains and abundant amino groups. When it was grafted onto magG@PDA, it strongly enhanced the hydrophilic properties of the magnetic composites. The PAMAM-functionalized magnetic composites were employed in the enrichment of glycopeptides, 15 glycopeptides from horseradish peroxidase (HRP) digestion were identified and the limit of detection was as low as 1 fmol μL−1. Also, it showed a good selectivity when the background nonglycopeptides had a concentration 100 fold higher than the target glycopeptides. All the results proved that magG@PDA@PAMAM has great potential in the glycoproteome analysis of complex biological samples.

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.Keywords: boronic-acid-functionalized magnetic graphene; enrichment; glycoproteome; MALDI-TOF mass spectrometry; phenolic-formaldehyde resin;

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.Keywords: circulating tumor cells; large-scale surface-enhanced Raman scattering imaging; nitrocellulose membrane substrate;

In our work, a new extraction tip with gold-modified polymer is developed. The simple, self-made and extremely economical tips were successfully applied to capture cysteine-containing peptides. The loading capacity of a tip (column bed: 0.3 mm diameter, 5 mm length) is 2–4 μg peptides. We can make one tip in 30 s and each costs less than 0.1 cent. The use of these tips can achieve a stable analysis with less background interference, even for 10 ng target peptides. Compared with other separation techniques, our method can save much time and energy while providing a means to selectively capture cysteine-containing peptides from complex analyte due to the strong interaction. All results showed that our new extraction tips have minimal cost and perfect selectivity; thus they have great potential in sample pretreatment systems for proteomics.

In this report, laser radiation (808 nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60 s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.Highlights► The near-IR laser radiation can accelerate the proteolysis efficiency through IMER significantly. ► The whole process can be completed in 1 min and room temperature. ► Satisfying analysis of human serum sample using laser-assisted IMER proteolysis.

In recent MS-based glycoproteomics, the selective enrichment of glycopeptides from complex biological samples is essential. In this work, for the first time, a novel ultra hydrophilic dendrimer-modified magnetic graphene@polydopamine@poly(amidoamine) (magG@PDA@PAMAM) was synthesized via three simple and rapid steps. The magnetic composites combined large surface of graphene, strong magnetic responsiveness of Fe3O4 with double hydrophilic abilities of PDA and PAMAM. Especially PAMAM had long dendritic chains and abundant amino groups. When it was grafted onto magG@PDA, it strongly enhanced the hydrophilic properties of the magnetic composites. The PAMAM-functionalized magnetic composites were employed in the enrichment of glycopeptides, 15 glycopeptides from horseradish peroxidase (HRP) digestion were identified and the limit of detection was as low as 1 fmol μL−1. Also, it showed a good selectivity when the background nonglycopeptides had a concentration 100 fold higher than the target glycopeptides. All the results proved that magG@PDA@PAMAM has great potential in the glycoproteome analysis of complex biological samples.