Selective enrichment of peptides from complex biosamples is essential for mass spectrometry-based proteomics but still remains a challenge. In this work, a facile approach was developed for rapid room-temperature synthesis of core–shell structured magnetic covalent organic framework composite nanospheres (denoted as Fe3O4@TbBd) by using monodisperse Fe3O4 nanoparticles as the magnetic core and 1,3,5-triformylbenzene (Tb) and benzidine (Bd) as two building blocks in the presence of dimethyl sulfoxide (DMSO). The resultant core–shell structured Fe3O4@TbBd nanospheres exhibited high adsorption capacity (28.5 mg g−1), fast adsorption kinetics (<5 min) and excellent reusability (more than 30 times) for peptides, owing to their specific properties of high surface area (196.21 m2 g−1), large pore volume (0.63 cm3 g−1), narrow pore size distribution (∼2.8 nm), strong magnetic response (41.5 emu g−1), as well as good thermal and chemical stability. Moreover, the Fe3O4@TbBd nanospheres also showed good selectivity towards hydrophobic peptides and a size-exclusion effect against proteins due to the inherent π–π stacking interaction and interconnected mesoporous channels of covalent organic framework (COF) shells. Taking advantage of these composite nanospheres, selective extraction and efficient enrichment of low abundance hydrophobic peptides from human serum in the presence of high abundance proteins were achieved. Based on the HPLC-Q-TOF/MS results, 29 hydrophobic peptides assigned to 12 proteins were clearly identified in 5 ng μL−1 human serum digestion upon treatment with Fe3O4@TbBd nanospheres, much better than those obtained without treatment, confirming the outstanding performance of the Fe3O4@TbBd nanospheres in proteome analysis.

Core–shell structured magnetic covalent organic frameworks (Fe3O4@COFs) were synthesized via a facile approach at room temperature. Combining the advantages of high porosity, magnetic responsiveness, chemical stability and selectivity, Fe3O4@COFs can serve as an ideal absorbent for the highly efficient enrichment of peptides and the simultaneous exclusion of proteins from complex biological samples.

Artificial enzyme mimetics have received considerable attention because natural enzymes have some significant drawbacks, including enzyme autolysis, low catalytic activity, poor recovery, and low stability to environmental changes. Herein, we demonstrated a facile approach for one-pot synthesis of hemeprotein-metal organic framework hybrid composites (H-MOFs) by using bovine hemoglobin (BHb) and zeolitic imidazolate framework-8 (ZIF-8) as a model reaction system. Surprisingly, the new hybrid composites exhibit 423% increase in peroxidase-like catalytic activity compared to free BHb. Taking advantages of the unique pore structure of H-MOFs with high catalytic property, a H-MOFs-based colorimetric biosensing platform was newly constructed and applied for the fast and sensitive detection of hydrogen peroxide (H2O2) and phenol. The corresponding detection limits as low as 1.0 μM for each analyte with wide linear ranges (0–800 μM for H2O2 and 0–200 μM for phenol) were obtained by naked-eye visualization. Significantly, a sensitive and selective method for visual assay of trace H2O2 in cells and phenol in sewage was achieved with this platform. The stability of H-MOFs was also examined, and excellent reproducibility and recyclability without losing in their activity were observed. In addition, the general applicability of H-MOFs was also investigated by using other hemeproteins (horseradish peroxidase, and myoglobin), and the corresponding catalytic activities were 291% and 273% enhancement, respectively. This present work not only expands the application of MOFs but also provides an alternative technique for biological and environmental sample assay.Keywords: colorimetric detection; hemeproteins; hydrogen peroxide; metal−organic framework; peroxidase mimetics; phenol

This work reports a novel metal–organic framework (MOF)-based metal affinity platform for the rapid and highly specific separation of histidine-rich proteins using zeolitic imidazolate framework-8 coated magnetic nanocomposites (denoted as Fe3O4@ZIF-8). The coating of the ZIF-8 layer on the Fe3O4 core was performed in aqueous solution at room temperature and merely took 10 minutes. The monodisperse Fe3O4@ZIF-8 has an average diameter of 190 nm, displays superparamagnetism with a saturation magnetization value of 47.9 emu g−1, and possesses a large external surface area of 131.0 m2 g−1. Due to the high density of low-coordinated Zn atoms on the surface of ZIF-8, Fe3O4@ZIF-8 exhibited a large adsorption capacity for model histidine-rich proteins (>6000 mg g−1 for bovine hemoglobin) and relatively low adsorption capacities for other proteins which contain fewer surface-exposed histidine residues. Moreover, Fe3O4@ZIF-8 showed excellent recyclability (more than 10 times) with high recovery (88.4%). In addition, Fe3O4@ZIF-8 can be used to selectively separate hemoglobin from a protein mixture and human blood samples. The good results demonstrate the potential of Fe3O4@ZIF-8 in the separation of histidine-rich proteins.

A facile approach for the synthesis of enzyme–inorganic hybrid nanoflowers and their application as an immobilized α-chymotrypsin (ChT) reactor (IMER) for highly efficient protein digestion was described. The hybrid nanoflowers were room-temperature synthesized in aqueous solution using calcium phosphate (Ca3(PO4)2) as the inorganic component and ChT as the organic component. The effects of reaction parameters on the formation of the enzyme-embedded hybrid nanoflowers and their growth mechanism were investigated systematically. By monitoring the reaction of N-benzoyl-L-tyrosine ethyl ester (BTEE), the enzymatic activity of the immobilized ChT was calculated and the results showed 266% enhancement in enzymatic activity. The performance of such a nanoreactor was further demonstrated by digesting bovine serum albumin (BSA) and human serum albumin (HSA), with a stringent threshold for unambiguous identification of these digests, the yielding sequence coverages for nanoflower-based digestion were 48% and 34%, higher than those obtained with the free enzyme. The digestion time of BSA and HSA in the former case was less than 2 min, about 1/360 of that performed in the latter case (12 h). Furthermore, the residual activity of the nanoflowers decreased slightly even after eight repeated use, demonstrating promising stability. In addition, the hybrid nanoflower-based IMER was applicable to the digestion of a complex human sample, showing great promise for proteome analysis.

Selective separation of intact phosphoproteins from complex biological samples is essential for ongoing, top-down phosphoproteomics but challenges still remain. Herein, aluminium glycinate functionalized silica nanoparticles (denoted as AGNP) were synthesized by a facile approach and applied for the specific capture of phosphoproteins. The selectivity and binding capacity of AGNP were evaluated using caseins (α-casein and β-casein) as phosphoproteins and bovine haemoglobin, bovine serum albumin, horseradish peroxidase, myoglobin and lysozyme as nonphosphoproteins. The results indicated that AGNP showed high binding capacity and selectivity for phosphoproteins (α-casein 1190 mg g−1 and β-casein 1060 mg g−1). In addition, AGNP was used to selectively capture and enrich phosphoproteins from protein mixtures and drinking milk samples. The good results demonstrate the potential of AGNP in phosphoproteomics analysis.

Combining free radical polymerization with click chemistry via a copper-mediated azide/alkyne cycloaddition (CuAAC) reaction in a “one-pot” process, a facile approach was developed for the preparation of a poly(3′-azido-3′-deoxythymidine-co-propargyl methacrylate-co-pentaerythritol triacrylate) (AZT-co-PMA-co-PETA) monolithic column. The resulting poly(AZT-co-PMA-co-PETA) monolith showed a relatively homogeneous monolithic structure, good permeability and mechanical stability. Different ratios of monomers and porogens were used for optimizing the properties of a monolithic column. A series of alkylbenzenes, amides, anilines, and benzoic acids were used to evaluate the chromatographic properties of the polymer monolith in terms of hydrophobic, hydrophilic and cation-exchange interactions, and the results showed that the poly(AZT-co-PMA-co-PETA) monolith exhibited more flexible adjustment in chromatographic selectivity than that of the parent poly(PMA-co-PETA) and AZT-modified poly(PMA-co-PETA) monoliths. Column efficiencies for toluene, DMF, and formamide with 35000–48000 theoretical plates per m could be obtained at a linear velocity of 0.17 mm s−1. The run-to-run, column-to-column, and batch-to-batch repeatabilities of the retention factors were less than 4.2%. In addition, the proposed monolith was also applied to efficient separation of sulfonamides, nucleobases and nucleosides, anesthetics and proteins for demonstrating its potential.

CuFe2O4 magnetic nanocrystal clusters (CuFe2O4 MNCs) were proposed as a new matrix for small molecule analysis by negative ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the first time. We demonstrated its advantages over conventional organic matrices in the detection of small molecules such as amino acids, peptides, nucleobases, fatty acids, and steroid hormones. A systematic comparison of CuFe2O4 MNCs with different ionization modes revealed that MS spectra obtained for the CuFe2O4 MNC matrix in the negative ion mode was only featured by deprotonated ion peaks with a free matrix background, which was different from the complicated alkali metal adducts produced in the positive ion mode. The developed method was found relatively tolerant to salt contamination and exhibited good reproducibility. A detection limit down to the subpicomolar level was achieved when testosterone was analyzed. In addition, by comparison of the MS spectra obtained from bare Fe3O4 and MFe2O4 MNC (M = Co, Ni, Cu, Zn) matrices, two main factors of MFe2O4 MNC matrices were revealed to play a vital role in assisting the negative ion desorption/ionization (D/I) process: doping transition metals into ferrite nanocrystals favoring laser absorption and energy transfer and a good match between the UV absorption of MFe2O4 MNCs and the excitation of nitrogen laser source facilitating LDI efficiency. This work creates a new branch of application for MFe2O4 MNCs and provides an alternative solution for small molecule analysis.

The application of the metal–organic framework (MOF) MIL-53(Fe) as a novel stationary phase for reverse-phase high performance liquid chromatography (HPLC) separation of positional isomers is described for the first time. Under the optimized conditions, baseline separation of xylene, dichlorobenzene, chlorotoluene and nitroaniline isomers was achieved on the MIL-53(Fe) packed column within a short time. The retention mechanisms of these isomers on the MIL-53(Fe) packed column were discussed in detail, and a typical reverse-phase behavior can describe the results. The thermodynamic characteristics of the HPLC separation of positional isomers were also evaluated. It was confirmed that the HPLC separation of positional isomers was controlled by the Gibbs free energy change (ΔG). In addition, reverse-phase HPLC separation of the tested isomers was also carried on MIL-53(Al, Cr) packed columns to investigate the influence of the metal centre of the MIL-53 framework on their chromatographic behaviors. The results showed that the MIL-53(Fe) packed column exhibited better separation performance than the MIL-53(Al, Cr) packed columns. Moreover, the MIL-53(Fe) packed column also gave efficient HPLC separation of alkylbenzene and naphthalene, aniline compounds and polycyclic aromatic hydrocarbons (PAHs). These successful applications suggest the potential of MIL-53(Fe) as a novel stationary phase for efficient reverse-phase HPLC separation.

Combining free radical polymerization with click chemistry via copper-mediated azide–alkyne cycloaddition (CuAAC) reaction in a “one-pot” process, a facile approach was developed for the preparation of a polymer monolithic column carrying 6-azidohexanoic acid (AHA), which was applied in capillary liquid chromatography (cLC) separations in the mixed-mode. The synthetic procedure was simple, time-saving and without any post modification. The morphology, permeability, and pore properties of the prepared polymer monolithic columns were characterized and the results showed the polymer monoliths have a uniform monolithic structure with high porosity. The retention behavior of the neutral and polar solutes on the polymer monoliths confirmed the successful incorporation of ligand in the monolithic matrix. A series of alkylbenzenes, phenols, and anilines were used to evaluate the chromatographic properties of the polymer monolith in terms of hydrophobic, hydrophilic and cation-exchange interactions. In addition, the poly (AHA-co-propargyl methacrylate-co-ethylene dimethacrylate) (AHA-co-PMA-co-EDMA) monolithic column could be successfully applied to the separation of polycyclic aromatic hydrocarbons (PAHs), nucleosides and nucleobases, alkaloids, peptides and proteins. The reproducibility of the monolithic column was less than 4.0% in terms of relative standard deviation (RSD) of the retention factor. The results demonstrate that the proposed method was an effective alternative for the preparation of the polymer monolith.

A facile strategy based on the synergistic effect of molecular imprinting and boronate affinity was proposed for glycoprotein imprinting. Polydopamine (PDA)-coated boronic acid-functionalized molecularly imprinted silica nanoparticles (MIPs) were prepared by the “thiol–ene” click reaction using SiO2 as the core, 3-acrylamidophenyl boronic acid (AAPBA) as the functional monomer, and horseradish peroxidase (HRP) as the glycoprotein template. A well defined core–shell structure of MIPs was obtained after self-polymerization of dopamine (DA) on the surface of HRP-immobilized silica nanoparticles (NPs). The polymerization conditions and adsorption behavior were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the HRP-MIPs showed higher binding affinity towards HRP than non-imprinted nanoparticles (NIPs), and the corresponding adsorption capacity (Q) and imprinted factor (α) reached 0.58 μmol g−1 and 2.6, respectively. The specificity for HRP recognition was evaluated with a competitive experiment, and the results indicated that the HRP-MIPs had higher selectivity for the template. The good features of the HRP-MIPs facilitated selective isolation and enrichment of trace HRP from human serum. In addition, the stability and regeneration were also investigated, which indicated that the HRP-MIPs had excellent reusability.

This work reports a facile ligand-free method for the rapid and highly specific separation of histidine (His)-rich proteins using CuFe2O4 magnetic nanocrystal clusters (MNCs). Monodispersed CuFe2O4 MNCs were synthesized via a simple and economical one-pot hydrothermal process. The resulting MNCs were characterized in detail. The measurements indicated that the MNCs exhibited good dispersion, high crystallinity, and superparamagnetic properties. Moreover, the obtained MNCs had a high saturation magnetization (45.1 emu g−1), which was sufficient to accomplish fast and efficient separation with an external magnetic field. The selectivity and binding capacity of CuFe2O4 MNCs were evaluated using a His-rich protein (bovine haemoglobin) and other proteins (bovine serum albumin, human serum albumin, myoglobin, lysozyme, cytochrome c and horseradish peroxidase) containing fewer surface-exposed His residues as model samples. The most distinct feature of the CuFe2O4 MNCs is the high haemoglobin binding capacity (4475 mg g−1) due to the coordination between copper(II) ions and surface-exposed histidine resides of haemoglobin. In addition, the CuFe2O4 MNCs can be successfully employed to selectively bind and remove abundant haemoglobin from human blood samples. The good results demonstrate the potential of CuFe2O4 MNCs in the separation of His-rich proteins.

A novel imprinting strategy using reversible covalent complexation of glycoprotein is described for creating glycoprotein-specific recognition cavities on 3-acrylamidophenylboronic acid-immobilized silica nanoparticles (SiO2@AAPBA). Two kinds of organic silanes (3-aminopropyltriethoxysilane (APTES) and n-octyltrimethoxysilane (OTMS)) were then polymerized on the surface of SiO2@AAPBA after the template (horseradish peroxidase (HRP)) was covalently immobilized by forming cyclic boronate complexes and their influence was examined. The results showed that not only the silane composition but also the relative proportions play an important role in glycoprotein imprinting. The template recognition properties were evaluated by single-protein or competitive batch rebinding experiments, and the results showed that the HRP-imprinted silica nanoparticles (HRP-MIP silica NPs) exhibited higher recognition ability and selectivity towards the template than the nonimprinted silica NPs and their corresponding imprinted factor (a) reached 2.71. The as-prepared HRP-MIP silica NPs could not only differentiate the template from another glycoprotein, but also enrich HRP from spiked human serum. The good results demonstrated their potential in glycoproteomic analysis.

•A novel silica-UIO-66 composite was synthesized for liquid chromatographic stationary phase.•Fast separation of ethylbenzene and styrene was achieved with the packed column.•Baseline separation of positional isomers was achieved with the packed column.A silica-UIO-66 composite was fabricated by a simple hydrothermal method and then applied as liquid chromatographic stationary phase for fast and efficient separation. X-ray diffraction patterns showed the presence of UIO-66 crystals in the silica-UIO-66 composites; while scanning electron microscope (SEM) images revealed that silica-UIO-66 composites were a homogeneous mixture of silica bead and UIO-66 crystals. A variety of substituted aromatics, chlorobenzene compounds and polycyclic aromatic hydrocarbons (PAHs) were used to evaluate the retention properties of the silica-UIO-66 composite packed column. Under the optimized conditions, baseline separation of ethylbenzene (EB) and styrene was obtained with high resolution and short retention time. In addition, the silica-UIO-66 composite packed column also showed some advantages in separation of positional isomers, with which baseline separation of EB and xylene, chlorotoluene and dichlorobenzene isomers was achieved. Moreover, the retention mechanisms of these compounds were also discussed in detail. The relative standard deviations (RSDs) for the separation of EB and xylene, chlorotoluene and dichlorobenzene isomers, as well as EB and styrene were 0.42–0.9%, 1.0–1.9%, 0.75–2.0%, and 0.9–2.1% for the retention time, peak area, peak height, and half peak width, respectively. The column efficiencies for EB, p-chlorotoluene, p-dichlorobenzene and styrene were 8780, 9060, 9990 and 5130 plates/m. The successful applications suggested high potentials of silica-MOFs composite as stationary phase for fast and efficient liquid chromatography separation.

•Hydrophilic magnetic mesoporous nanoparticles were synthesized via click chemistry.•The mesoporous nanoparticles showed high hydrophilicity and large surface area.•Successful application to the selective enrichment of glycopeptides from protein digests.•Successful application to the selective enrichment of glycans from digested human serum.Selective enrichment of glycopeptides from complex biological samples is essential for MS-based glycoproteomics, but challenges still remain. In this work, glucose-functionalized magnetic mesoporous nanoparticles (MMNs), which hold the attractive features of well-defined core/shell structure, high specific surface area (324 m2/g), narrow pore size distribution (2.2 nm) and high magnetic responsivity (69.1 emu g−1), were synthesized via click chemistry and applied to enrich glycopeptides and glycans. Taking advantages of the size-exclusive effect of mesopore against proteins and the hydrophilic interaction between glycans and glucose, the hydrophilic MMNs possessed high selectivity for glycopeptides at the digested mixtures of horseradish peroxidase (HRP), myoglobin and β-casein at molar ratio of 1:1:10, large enrichment capacity (as high as 250 mg/g), high sensitivity (50 fmol), excellent speed (5 min for enrichment) and high recovery of glycopeptides (as high as 94.6%). Additionally, the MMNs exhibited excellent performance in enrichment of N-linked glycans from the digested human serum that are made up of peptides, large proteins and other compounds. These outstanding features will give the hydrophilic MMNs high benefit for MS analysis of low-abundance glycopeptides/glycans.

•A GSH–silica hybrid monolith was prepared via thiol-ene click reaction in one-pot process.•The obtained hybrid monolith showed a mixed-mode chromatographic behavior.•It can be applied to the separation of a series of small molecules and protein tryptic digests.A novel glutathione (GSH)–silica hybrid monolithic column synthesized via a combination of thiol-ene click reaction and one-pot process was described, where thiol-end GSH organic monomer and 2,2-azobisisobutyronitrile (AIBN) were mixed with hydrolyzed tetramethyloxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) and then introduced into a fused-silica capillary for simultaneous polycondensation and “thiol-ene” click reaction to form the GSH–silica hybrid monolith. The effects of the molar ratio of TMOS/γ-MAPS, the amount of GSH, and the volume of porogen on the morphology, permeability and pore properties of the prepared GSH–silica hybrid monoliths were studied in detail. A uniform monolithic network with high porosity was obtained. A series of test compounds including alkylbenzenes, amides, and anilines were used to evaluate the retention behaviors of the GSH–silica hybrid monolithic column. The results demonstrated that the prepared GSH–silica hybrid monolith exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange interaction. The run-to-run, column-to-column and batch-to-batch reproducibilities of the GSH–silica hybrid monolith for phenols’ retention were satisfactory with the relative standard deviations (RSDs) less than 1.3% (n = 5), 2.6% (n = 3) and 3.2% (n = 3), respectively, indicating the effectiveness and practicability of the proposed method. In addition, the GSH–silica hybrid monolith was applied to the separation of nucleotides, peptides and protein tryptic digests, respectively. The successful applications suggested the potential of the GSH–silica hybrid monolith in complex sample analysis.

A novel kind of immobilized trypsin reactor based on enzyme–inorganic hybrid nanoflowers was first developed and can be applied to ultra fast, highly efficient proteome digestion.

A phenylboronic acid-silica hybrid monolithic column for capillary liquid chromatography (cLC) was prepared through one-pot process by using 4-vinylphenylboronic acid (VPBA) and alkoxysilanes simultaneously. The effects of the molar ratio of tetramethyloxysilane/γ-methacryloxypropyltrimethoxysilane (TMOS/γ-MAPS), amount of VPBA, and the volume of diethylene glycol (DEG) on the morphologies, permeabilities and pore properties of the prepared VPBA-silica hybrid monolithic columns were studied in detail. A relatively uniform monolithic structure with high porosity was obtained with optimized ingredients. A series of cis-diol-containing compounds, alkylbenzenes, amides, and anilines were utilized to evaluate the retention behaviors of the VPBA-silica hybrid monolithic column. The result demonstrated that the prepared VPBA-silica hybrid monolithic column exhibited multiple interactions including hydrophobicity, hydrophilicity, as well as cation exchange apart from the expected affinity interaction. The run-to-run, column-to-column and batch-to-batch reproducibility of the VPBA-silica hybrid monolith were satisfactory with the relative standard deviations (RSDs) less than 1.63% (n = 5), 2.02% (n = 3) and 2.90% (n = 5), respectively, indicating the effectiveness and practicability of the proposed method. In addition, the VPBA-silica hybrid monolithic column was further applied to the separation of proteins and tryptic digest of bovine serum albumin (BSA), respectively. The successful applications suggested the potential of the VPBA-silica hybrid monolith in proteome analysis.Highlights► A facile one-pot approach was developed for preparation of VPBA-silica hybrid monolith. ► The obtained monolith showed a mixed-mode retention behavior. ► It can be applied to the separation of small molecules, tryptic digest and proteins.

•A facile approach was developed for preparation of glycoprotein-imprinted monolithic column.•The as-prepared imprinted monolith showed high affinity toward template glycoprotein.•It was successfully applied to the selective enrichment of HRP from human serum sample.A novel imprinting strategy using reversible covalent complexation of glycoprotein was described for creating glycoprotein-specific recognition cavities on boronate-functionalized monolithic column. Based on it, a molecularly imprinted monolithic column was prepared by self-polymerization of dopamine (DA) on the surface of 4-vinylphenylboronic acid (VPBA)-based polymeric skeletons after reversible immobilization of horseradish peroxidase (HRP). Due to the combination of boronate affinity and surface imprinting of DA, the stable and accessible recognition sites in the as-prepared imprinted monolith could be obtained after the removal of the template, which facilitated the rebinding of the template and provided good reproducibility and lifetime of use. The recognition behaviors of proteins on the bare VPBA-based, HRP-imprinted and nonimprinted monolithic columns were evaluated in detail and the results showed that the HRP-imprinted monolith exhibited higher recognition ability toward the template than another two monolithic columns. Not only nonglycoproteins but also glycoproteins can be well separated with the HRP-imprinted monolith. In addition, the feasibility of the HRP-imprinted monolith, adopted as an in-tube solid phase microextraction (in-tube SPME), was further assessed by selective extraction and enrichment of HRP from human serum. The good results demonstrated its potential in glycoproteome analysis.

Dynamic coating of the surface in capillary electrophoresis with chemiluminescence detection (CE-CL) using an off-column coaxial flow interface for the determination of four hemeproteins was developed. This method is based on the luminol–hydrogen peroxide reaction catalyzed by metalloproteins in alkaline medium. The experimental setup of the CE-CL system with the proposed off-column coaxial interface was evaluated by separation and detection of dopamine and catechol based on inhibition of the luminol–potassium ferricyanide reaction. Highly efficient separation of the two model compounds with symmetrical peak shape and satisfactory reproducibility was achieved by using this interface. In addition, in order to obtain a good resolution for hemeproteins, polyvinylpyrrolidone (PVP) combined with sodium dodecyl sulfate (SDS) were introduced as dynamic modifiers to reduce the unwanted adsorption of non-specific protein. Several parameters affecting the CE separation and CL detection were investigated in detail. Under the optimized conditions, a mixture of the four hemeproteins (horseradish peroxidase (HRP), catalase (Cat), myoglobin (Mb) and cytochrome C (Cyt C)) could be well separated within 20 min. The linear ranges of the four proteins were 5.7 × 10−8 to 1.1 × 10−6 mol L−1 for HRP, 4.0 × 10−8 to 2.0 × 10−6 mol L−1 for Cat, 1.1 × 10−10 to 5.6 × 10−8 mol L−1 for Mb, and 3.8 × 10−7 to 7.7 × 10−6 mol L−1 for Cyt C. The limits of detection (LODs) (S/N = 3) for HRP, Cat, Mb and Cyt C were 2.2 × 10−8 mol L−1 (104.5 amol), 1.6 × 10−8 mol L−1 (74 amol), 5.6 × 10−11 mol L−1 (0.26 amol), and 1.95 × 10−7 mol L−1 (0.89 fmol), respectively. The proposed method has been successfully applied to the analysis of low-level Mb in a spiked human urine sample and the recoveries were above 97%. Our primary result demonstrated that the proposed CE-CL method has great potential for Mb determination in clinical diagnosis.

A molecularly imprinted inorganic–organic hybrid monolithic capillary column (MIP hybrid monolith) was synthesized by one-pot process and its application in selective recognition and capture of lysozyme (Lyz) from complex biological samples was described for the first time. Due to a combination of rigid silica matrices and flexible organic hydrogels in one-pot process, stable and accessible recognition sites in the as-prepared MIP hybrid monolith could be obtained after the removal of template protein, which facilitated the rebinding of template and provided good reproducibility and lifetime of use. The morphology, permeability, and pore properties of the as-prepared MIP hybrid monolith were characterized and a uniform monolithic matrix with high surface area and large through-pores was observed. The recognition behavior of MIP and non-imprinted (NIP) hybrid monolith was evaluated by separating template protein from unfractionated protein mixture and the result indicated that the MIP hybrid monolith has much higher affinity toward the template protein than NIP hybrid monolith. High imprinted factor (IF) and separation efficiency could be obtained. In addition, the practicality of the Lyz-MIP hybrid monolith was further evaluated by selective separation of Lyz from egg white and capture of Lyz from human serum by adopting it as an in-tube solid phase microextraction (in-tube SPME), and the good results demonstrated its potential in proteome analysis.Highlights► A facile one-pot approach was developed for preparation of protein-imprinted hybrid monolith. ► The obtained monolith showed high selectivity toward template protein. ► It can be applied to the selective isolation and capture of Lyz from complex biological samples.

•A facile method was developed for synthesis of PDA-coated protein-imprinted silica NPs.•The imprinted silica NPs showed large binding capacity and high selectivity toward template.•The imprinted silica NPs can be applied to the depletion of high-abundance BHb from cattle blood.Surface imprinting over nanostructured matrices is an effective solution to overcome template removal and achieve high binding capacity. In this work, a facile method was developed for synthesis of polydopamine-coated molecularly imprinted silica nanoparticles (PDA-coated MIP silica NPs) based on self-polymerization of dopamine (DA) on the surface of silica NPs in the presence of template protein. Transmission electronic microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR) and thermogravimetric analysis (TGA) showed that PDA layers were successfully attached on the surface of silica NPs and the corresponding thickness was about 5 nm, which enabled the MIP silica NPs to have fast binding kinetics and high binding capacity. Under the aqueous media, the imprinted silica NPs showed much higher binding affinity toward template than non-imprinted (NIP) silica NPs. The protein recognition properties were examined by single-protein or competitive batch rebinding experiments and rebinding kinetics study, validating that the imprinted silica NPs have high selectivity for the template. The resultant BHb–MIP silica NPs could not only selectively separate BHb from the protein mixture, but also specifically deplete high-abundance BHb from cattle whole blood. In addition, the stability and regeneration were also investigated, which indicated that the imprinted silica NPs had excellent reusability.

Combining surface imprinting with nanomaterials is an effective solution to overcome template removal and achieve large binding capacity. In this work, highly monodisperse and uniform-sized silica nanoparticles (NPs) with average diameter of ∼400 nm were synthesized by using tetraethoxysilane (TEOS) as a single precursor, and then vinyl groups were introduced onto the surface of silica NPs by chemical modification of γ-methacryloxypropyltrimethoxysilane (γ-MAPS). Subsequently, the molecularly imprinted polymer (MIP) coating was copolymerized and anchored onto the surface of vinyl modified silica NPs dispersed in aqueous media with lysozyme (Lyz) as a template. The morphology and structure property of the resultant MIP-coated silica NPs were characterized by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR). The polymerization and adsorption conditions were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the imprinted nanoparticles showed higher binding affinity toward the template than non-imprinted (NIP) nanoparticles, and the corresponding imprinted factor (a) reached 1.68. The specificity for Lyz recognition was evaluated with competitive experiments, indicating the imprinted nanoparticles have a higher selectivity for the template. The resultant Lyz-MIP silica NPs could not only selectively extract a target protein from mixed proteins, but also specifically enrich Lyz from human serum. In addition, the stability and regeneration were also investigated, which indicated the imprinted silica NPs had excellent reusability.

A new polymer monolith with three modes of reverse-phase, hydrophilic and cation-exchange interaction was synthesized in 100 μm i.d. fused-silica capillary by in situ polymerization procedure. The pre-polymerization mixture consisted of glycidyl methacrylate (GMA) and 4-vinylphenylboronic acid (VPBA) as bifunctional monomers, ethylene dimethacrylate (EDMA) as crosslinker, 1,4-butanediol (BDO) and diethylene glycol (DEG) as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The resulting poly(GMA-co-VPBA-co-EDMA) monolith showed a relatively homogeneous monolithic structure, good permeability and mechanical stability. Different ratios of monomers and porogens were used for optimizing the properties of monolithic column. The column performance was assessed by the separation of a series of neutral solutes, charge solutes, phenols and anilines. Compared with poly(GMA-co-EDMA) monolith, the proposed monolith exhibited more flexible adjustment of selectivity in terms of hydrophobic, hydrophilic, as well as cation-exchange interaction in the same chromatographic conditions. High column efficiencies for benzene derivatives with 70,000–102,000 theoretical plates/m could be obtained at a linear velocity of 0.265 mm/s. The run-to-run, column-to-column, and batch-to-batch repeatabilities of the retention times were less than 8.23%. Additionally, the purposed monolith was also applied to efficient separation of alkaloids and proteins for demonstrating its potential in biomolecule separation.Highlights► A new poly(GMA-co-VPBA-co-EDMA) monolithic column was synthesized. ► It can be applied to the separation of a series of small molecules and proteins. ► The purposed monolith exhibits high separation abilities and selectivity.

A novel method of microemulsion electrokinetic capillary chromatography (MEEKC) coupled with on-line large volume sample stacking was developed for the analysis of six plant hormones including indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, 1-naphthaleneacetic acid, abscisic acid and salicylic acid. Baseline separation of six plant hormones was achieved within 10 min by using the microemulsion background electrolyte containing a 97.2% (w/w) 10 mM borate buffer at pH 9.2, 1.0% (w/w) ethyl acetate as oil droplets, 0.6% (w/w) sodium dodecyl sulphate as surfactant and 1.2% (w/w) 1-butanol as cosurfactant. In addition, an on-line concentration method based on a large volume sample stacking technique and multiple wavelength detection was adopted for improving the detection sensitivity in order to determine trace level hormones in a real sample. The optimal method provided about 50–100 fold increase in detection sensitivity compared with a single MEEKC method, and the detection limits (S/N = 3) were between 0.005 and 0.02 μg mL−1. The proposed method was simple, rapid and sensitive and could be applied to the determination of six plant hormones in spiked water samples, tobacco leaves and 1-naphthylacetic acid in leaf fertilizer. The recoveries ranged from 76.0% to 119.1%, and good reproducibilities were obtained with relative standard deviations (RSDs) less than 6.6%.

A mixed-mode monolithic stationary phase was prepared for capillary liquid chromatography (cLC) by in situ copolymerization of 4-vinylphenylboronic acid (VPBA) and pentaerythritol triacrylate (PETA) in a binary porogenic solvent consisting of ethylene glycol/cyclohexanol. The monomer of VPBA functioned as ion-exchange sites, hydrophilic ligands, hydrophobic groups and affinity sites, while PETA was introduced as a hydrophilic crosslinker. The resultant monoliths with different column properties (e.g. morphology, permeability and selectivity) were optimized by adjusting the ratio of VPBA to PETA and the composition of porogenic solvent. The results showed that the selectivity of the monoliths increased with increasing content of VPBA in the polymerization mixture. A series of alkylbenzenes, amides, and anilines were used to evaluate the column performance in terms of hydrophobic, hydrophilic and cation-exchange interactions. At an optimized flow rate of 50 μL/min (corresponding to 0.265 mm/s), the monolith exhibited high column efficiencies of 43,000–100,000 plates/m for alkylbenzenes. Good repeatability was obtained with relative standard deviation (RSD) of retention factor (k) less than 0.65% for run-to-run (n = 5) and less than 2.49% for column-to-column (n = 5). In addition, the poly(VPBA-co-PETA) monolithic column was applied to the separation of phenols, nucleobases, and proteins, respectively. These successful applications demonstrate the purposed monoliths are promising for cLC separation of small molecules and proteins.Highlights► A mixed-mode poly(VPBA-co-PETA) monolithic column is prepared via single-step in situ polymerization. ► It can be applied to the separation of a series of small molecules and proteins. ► The purposed monolith exhibits high separation ability and selectivity.

A facile one-pot approach was developed for synthesis of phenylboronic acid-functionalized core-shell magnetic nanoparticles. The resulting magnetic nanocomposites demonstrated great potential in selective enrichment of glycoproteins.

An inorganic–organic hybrid affinity monolithic column was synthesized by a novel “one-pot” approach. The resulting hybrid affinity monoliths have potential applications in specific recognition and enrichment of glycoproteins.

A phenylboronate affinity monolith was prepared and applied to the selective capture of glycoproteins from unfractionated protein mixtures. The monolith was synthesized in a 100 μm i.d capillary by an in situpolymerization procedure using a pre-polymerization mixture consisting of 4-vinylphenylboronic acid (VPBA) as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, diethylene glycol and ethylene glycol as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The prepared monolith was characterized in terms of the morphology, pore property, and recognition property. The selectivity and dynamic binding capacity were evaluated by using standard glycoproteins and nonglycoproteins as model proteins. The chromatographic results demonstrated that the phenylboronate affinity monolith had higher selectivity and binding capacity for glycoprotein than nonglycoprotein. The resulting phenylboronate affinity monolith was used as the sorbent for in-tube solid phase microextraction (in-tube SPME), and the extraction performance of the monolith was assessed by capture of ovalbumin from egg white sample.

This study reports a facile approach for the synthesis of horseradish peroxidise (HRP)-inorganic hybrid nanoflowers by self-assembly of HRP and copper phosphate (Cu3(PO4)2·3H2O) in aqueous solution. Several reaction parameters that affect the formation of the hybrid nanoflowers were investigated and a hierarchical flowerlike spherical structure with hundreds of nanopetals was obtained under the optimum synthetic conditions. The enzymatic activity of HRP embedded in hybrid naonflowers was evaluated based on the principle of HRP catalyzing the oxidation of o-phenylenediamine (OPD) in the presence of hydrogen peroxide (H2O2). The results showed that 506% enhancement of enzymatic activity in the hybrid nanoflowers could be achieved compared with the free HRP in solution. Taking advantages of the structural feature with catalytic property, a nanoflower-based colorimetric platform was newly designed and applied for fast and sensitive visual detection of H2O2 and phenol. The limits of detection (LODs) for H2O2 and phenol were as low as 0.5 μM and 1.0 μM by the naked-eye visualization, which meet the requirements of detection of both analytes in clinical diagnosis and environmental water. The proposed method has been successfully applied to the analysis of low-level H2O2 in spiked human serum and phenol in sewage, respectively. The recoveries for all the determinations were higher than 92.6%. In addition, the hybrid nanoflowers exhibited excellent reusability and reproducibility in cycle analysis. These primary results demonstrate that the hybrid nanoflowers have a great potential for applications in biomedical and environmental chemistry.

Core–shell structured magnetic covalent organic frameworks (Fe3O4@COFs) were synthesized via a facile approach at room temperature. Combining the advantages of high porosity, magnetic responsiveness, chemical stability and selectivity, Fe3O4@COFs can serve as an ideal absorbent for the highly efficient enrichment of peptides and the simultaneous exclusion of proteins from complex biological samples.

An inorganic–organic hybrid affinity monolithic column was synthesized by a novel “one-pot” approach. The resulting hybrid affinity monoliths have potential applications in specific recognition and enrichment of glycoproteins.

A facile approach for the synthesis of enzyme–inorganic hybrid nanoflowers and their application as an immobilized α-chymotrypsin (ChT) reactor (IMER) for highly efficient protein digestion was described. The hybrid nanoflowers were room-temperature synthesized in aqueous solution using calcium phosphate (Ca3(PO4)2) as the inorganic component and ChT as the organic component. The effects of reaction parameters on the formation of the enzyme-embedded hybrid nanoflowers and their growth mechanism were investigated systematically. By monitoring the reaction of N-benzoyl-L-tyrosine ethyl ester (BTEE), the enzymatic activity of the immobilized ChT was calculated and the results showed 266% enhancement in enzymatic activity. The performance of such a nanoreactor was further demonstrated by digesting bovine serum albumin (BSA) and human serum albumin (HSA), with a stringent threshold for unambiguous identification of these digests, the yielding sequence coverages for nanoflower-based digestion were 48% and 34%, higher than those obtained with the free enzyme. The digestion time of BSA and HSA in the former case was less than 2 min, about 1/360 of that performed in the latter case (12 h). Furthermore, the residual activity of the nanoflowers decreased slightly even after eight repeated use, demonstrating promising stability. In addition, the hybrid nanoflower-based IMER was applicable to the digestion of a complex human sample, showing great promise for proteome analysis.

Selective separation of intact phosphoproteins from complex biological samples is essential for ongoing, top-down phosphoproteomics but challenges still remain. Herein, aluminium glycinate functionalized silica nanoparticles (denoted as AGNP) were synthesized by a facile approach and applied for the specific capture of phosphoproteins. The selectivity and binding capacity of AGNP were evaluated using caseins (α-casein and β-casein) as phosphoproteins and bovine haemoglobin, bovine serum albumin, horseradish peroxidase, myoglobin and lysozyme as nonphosphoproteins. The results indicated that AGNP showed high binding capacity and selectivity for phosphoproteins (α-casein 1190 mg g−1 and β-casein 1060 mg g−1). In addition, AGNP was used to selectively capture and enrich phosphoproteins from protein mixtures and drinking milk samples. The good results demonstrate the potential of AGNP in phosphoproteomics analysis.

A novel imprinting strategy using reversible covalent complexation of glycoprotein is described for creating glycoprotein-specific recognition cavities on 3-acrylamidophenylboronic acid-immobilized silica nanoparticles (SiO2@AAPBA). Two kinds of organic silanes (3-aminopropyltriethoxysilane (APTES) and n-octyltrimethoxysilane (OTMS)) were then polymerized on the surface of SiO2@AAPBA after the template (horseradish peroxidase (HRP)) was covalently immobilized by forming cyclic boronate complexes and their influence was examined. The results showed that not only the silane composition but also the relative proportions play an important role in glycoprotein imprinting. The template recognition properties were evaluated by single-protein or competitive batch rebinding experiments, and the results showed that the HRP-imprinted silica nanoparticles (HRP-MIP silica NPs) exhibited higher recognition ability and selectivity towards the template than the nonimprinted silica NPs and their corresponding imprinted factor (a) reached 2.71. The as-prepared HRP-MIP silica NPs could not only differentiate the template from another glycoprotein, but also enrich HRP from spiked human serum. The good results demonstrated their potential in glycoproteomic analysis.

Combining surface imprinting with nanomaterials is an effective solution to overcome template removal and achieve large binding capacity. In this work, highly monodisperse and uniform-sized silica nanoparticles (NPs) with average diameter of ∼400 nm were synthesized by using tetraethoxysilane (TEOS) as a single precursor, and then vinyl groups were introduced onto the surface of silica NPs by chemical modification of γ-methacryloxypropyltrimethoxysilane (γ-MAPS). Subsequently, the molecularly imprinted polymer (MIP) coating was copolymerized and anchored onto the surface of vinyl modified silica NPs dispersed in aqueous media with lysozyme (Lyz) as a template. The morphology and structure property of the resultant MIP-coated silica NPs were characterized by scanning electronic microscopy (SEM), transmission electronic microscopy (TEM) and Fourier transform infrared spectroscopy (FT-IR). The polymerization and adsorption conditions were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the imprinted nanoparticles showed higher binding affinity toward the template than non-imprinted (NIP) nanoparticles, and the corresponding imprinted factor (a) reached 1.68. The specificity for Lyz recognition was evaluated with competitive experiments, indicating the imprinted nanoparticles have a higher selectivity for the template. The resultant Lyz-MIP silica NPs could not only selectively extract a target protein from mixed proteins, but also specifically enrich Lyz from human serum. In addition, the stability and regeneration were also investigated, which indicated the imprinted silica NPs had excellent reusability.

This work reports a novel metal–organic framework (MOF)-based metal affinity platform for the rapid and highly specific separation of histidine-rich proteins using zeolitic imidazolate framework-8 coated magnetic nanocomposites (denoted as Fe3O4@ZIF-8). The coating of the ZIF-8 layer on the Fe3O4 core was performed in aqueous solution at room temperature and merely took 10 minutes. The monodisperse Fe3O4@ZIF-8 has an average diameter of 190 nm, displays superparamagnetism with a saturation magnetization value of 47.9 emu g−1, and possesses a large external surface area of 131.0 m2 g−1. Due to the high density of low-coordinated Zn atoms on the surface of ZIF-8, Fe3O4@ZIF-8 exhibited a large adsorption capacity for model histidine-rich proteins (>6000 mg g−1 for bovine hemoglobin) and relatively low adsorption capacities for other proteins which contain fewer surface-exposed histidine residues. Moreover, Fe3O4@ZIF-8 showed excellent recyclability (more than 10 times) with high recovery (88.4%). In addition, Fe3O4@ZIF-8 can be used to selectively separate hemoglobin from a protein mixture and human blood samples. The good results demonstrate the potential of Fe3O4@ZIF-8 in the separation of histidine-rich proteins.

A facile strategy based on the synergistic effect of molecular imprinting and boronate affinity was proposed for glycoprotein imprinting. Polydopamine (PDA)-coated boronic acid-functionalized molecularly imprinted silica nanoparticles (MIPs) were prepared by the “thiol–ene” click reaction using SiO2 as the core, 3-acrylamidophenyl boronic acid (AAPBA) as the functional monomer, and horseradish peroxidase (HRP) as the glycoprotein template. A well defined core–shell structure of MIPs was obtained after self-polymerization of dopamine (DA) on the surface of HRP-immobilized silica nanoparticles (NPs). The polymerization conditions and adsorption behavior were investigated in detail in order to obtain the highest selectivity and binding capacity. Under the optimized conditions, the HRP-MIPs showed higher binding affinity towards HRP than non-imprinted nanoparticles (NIPs), and the corresponding adsorption capacity (Q) and imprinted factor (α) reached 0.58 μmol g−1 and 2.6, respectively. The specificity for HRP recognition was evaluated with a competitive experiment, and the results indicated that the HRP-MIPs had higher selectivity for the template. The good features of the HRP-MIPs facilitated selective isolation and enrichment of trace HRP from human serum. In addition, the stability and regeneration were also investigated, which indicated that the HRP-MIPs had excellent reusability.

This work reports a facile ligand-free method for the rapid and highly specific separation of histidine (His)-rich proteins using CuFe2O4 magnetic nanocrystal clusters (MNCs). Monodispersed CuFe2O4 MNCs were synthesized via a simple and economical one-pot hydrothermal process. The resulting MNCs were characterized in detail. The measurements indicated that the MNCs exhibited good dispersion, high crystallinity, and superparamagnetic properties. Moreover, the obtained MNCs had a high saturation magnetization (45.1 emu g−1), which was sufficient to accomplish fast and efficient separation with an external magnetic field. The selectivity and binding capacity of CuFe2O4 MNCs were evaluated using a His-rich protein (bovine haemoglobin) and other proteins (bovine serum albumin, human serum albumin, myoglobin, lysozyme, cytochrome c and horseradish peroxidase) containing fewer surface-exposed His residues as model samples. The most distinct feature of the CuFe2O4 MNCs is the high haemoglobin binding capacity (4475 mg g−1) due to the coordination between copper(II) ions and surface-exposed histidine resides of haemoglobin. In addition, the CuFe2O4 MNCs can be successfully employed to selectively bind and remove abundant haemoglobin from human blood samples. The good results demonstrate the potential of CuFe2O4 MNCs in the separation of His-rich proteins.