•The first bullet point can be changed into: A fluorescence turn-on sensor for the sensitive detection of glycoproteins.•The limit of detection of the selected glycoproteins was lower than 0.1 nM.•The sensor could be applied in a wide pH range of 5.0–9.0.•High selectivity and anti-interference ability in TRF detection in serum.A simple fluorescence turn-on sensor has been designed for the highly sensitive detection of glycoproteins on the basis of boronic acid functional polymer capped Mn-doped ZnS quantum dots (QDs@MPS@AAPBA). In the absence of glycoproteins, the fluorescence emission intensity of the QDs@MPS@AAPBA was relatively weaker due to the effective electronic transfer from the QDs to the boron moieties on its surface. While the glycoproteins were introduced into the system, an obvious fluorescence enhancement was observed. It was attributed to the boron moieties covalent binding glycans of the glycoproteins resulting in the electronic transfer process being inhibited. Under the optimal conditions, this fluorescent probe not only could be applied in a wide pH range of 5.0–9.0, but also the binding constants and detection limits of the QDs@MPS@AAPBA for horseradish peroxidase (HRP) and transferrin (TRF) were up to 7.23 × 106 M−1, 1.53 × 107 M−1 and 1.44 × 10−10 M, 3.36 × 10−10 M, respectively. Finally, this proposed method has also been utilized for the TRF determination in serum without any complicated pretreatment and the recovery was in the range of 95.7%–103.0%. As a result, it is promising for application on the glycoproteins detection in complex biological samples.Download high-res image (185KB)Download full-size image

The development of methods to isolate and enrich low-abundance glycopeptides is an important prerequisite for glycoproteomics research. In this study, a hydrophilic maltose functionalized Au nanoparticle (NP)/polydopamine (PDA)/Fe3O4-reduced graphene oxide (RGO) nanocomposite has been successfully synthesized in mild conditions. The bioadhesive polydopamine film was prepared by self-polymerization on the surface of Fe3O4–graphene oxide, which not only prevents the agglomeration of the graphene sheets and enhances the specific surface area, but also facilitates the Au NP immobilization. A great number of loading Au NPs possess the highly available surface area for the immobilization of the high density of the thiol-terminated maltose via Au–S bonds. The resulting Au NP-maltose/PDA/Fe3O4-RGO nanocomposite exhibits excellent environmental stability, good biocompatibility and water dispersibility. Furthermore, the highly loaded Fe3O4 NPs make the enrichment very convenient. With all of these advances, the novel Au NP-maltose/PDA/Fe3O4-RGO nanocomposite presents selective enrichment of the glycopeptides from a low concentration of horseradish peroxidase tryptic digest (0.1 ng μL−1).

In this work, a novel kind of core–shell magnetic molecularly imprinted polymer (MIP) for sulfamethazine (SMZ) was synthesized by the surface-initiated atom transfer radical polymerization (ATRP) strategy. In this protocol, polydopamine was formed on the Fe3O4 nanoparticles (NPs) in 10 mM Tris–HCl buffer solution (pH 8.5). The initiator bromide reagent of ATRP was then grafted onto the polydopamine surface. Finally, the MIP layer was formed on the surface of Fe3O4 by the copolymerization of sulfamethazine as a template, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linking agent using an organometallic catalyst comprising Cu(I)Br and pentamethyldiethylenetriamine. The morphology and magnetic, adsorption and recognition properties of Fe3O4@SMZ-MIP NPs were characterized using transmission electron microscopy (TEM), Fourier transform infrared (FT-IR) spectroscopy, vibrating sample magnetometry (VSM), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and re-binding experiments. The controllable nature of ATRP allows the growth of a uniform MIP layer with adjustable thickness, providing a large adsorption capacity (680.27 μg g−1), fast kinetics about 40 min to equilibrium, and a considerably high imprinting factor of 17.02. The feasibility of the enrichment of sulfonamides by Fe3O4@SMZ-MIP was demonstrated using egg samples spiked with SMZ and SMR. The recoveries of SMZ and SMR ranged from 76.7 to 93.0% and 69.3 to 77.2%, respectively, and the relative standard deviations (RSD) were <7.0%. In addition, Fe3O4@SMZ-MIP showed good reusability for at least five repeated cycles.

Protein post-translational modifications (PTMs) play important roles in cellular physiology. Mass spectrometry (MS) has been developed into a powerful tool to identify all possible protein modifications. Herein, we describe our efforts to deduce the structures of two unknown modifications at tryptophan (Trp) residues (W + 92 Da and W + 108 Da). The two modifications were further confirmed by aligning the MS/MS fragmentation of synthetic peptide with in-vivo peptide identified. Finally, the mimic experiment elucidated how two Trp modifications occur. This study, therefore, expands current knowledge of Trp modifications.

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. In this paper, a facile and efficient approach combining distillation–precipitation polymerization (DPP) and click chemistry was developed to synthesize boronic acid ligand-modified magnetic nanoparticles for the enrichment of glycoproteins. Due to the relatively large amount of benzyl chloride groups introduced by DPP on the magnetic core, which easily can be transferred into azide groups, the alkyne–phenylboronic acid ligands were immobilized onto the surface of Fe3O4 with high efficiency via the Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click’ reaction. The morphology, structure and composition of the resulting core–shell Fe3O4@poly(4-vinylbenylchloride)@amidophenylboronic acid (Fe3O4@pVBC@APBA) nanocomposites were characterized by transmission electron microscopy, X-ray powder diffraction, vibrating sample magnetometry, Fourier transform infrared spectroscopy, thermogravimetric analysis and X-ray photoelectron spectrometry. The Fe3O4@pVBC@APBA microspheres held a ∼50 nm polymeric shell, and exhibited high magnetic response to an external magnetic field. The binding results demonstrated that Fe3O4@pVBC@APBA possessed high adsorption capacity and remarkable selectivity to glycoproteins. Moreover, the glycoproteins in the egg white sample could be enriched under physiological conditions (pH 7.4) as well, due to the lower pKa value of the alkyne–phenylboronic acid ligand. The high stability and selectivity of Fe3O4@pVBC@APBA for the glycoproteins were retained over several separation cycles. This boronate affinity material has potential applications in biomedical and biotechnological fields including drug delivery and biosensing.

A novel multifunctional graphene/Fe3O4/TiO2 composite with excellent hydrophilicity and biological compatibility was synthesized and applied to the fast, highly selective and sensitive enrichment of phosphopeptides from biosamples.

Microcystins (MCs), a family of potent cyclic heptapeptides, are produced by cyanobacteria blooms in eutrophic water and can cause acute and chronic toxicity and even mortality to animals and humans. Previous MC removal strategies concerned only highly contaminated water, in which the concentration of the pollutant was considerably larger than that in the natural world. Herein, we developed a ternary composite of TiO2-coated magnetic graphene and used it as an adsorbent and photocatalyst to efficiently remove microcystin-LR (MC-LR) from water. The two-dimensional sheets of graphene were decorated with a large quantity of spherical Fe3O4 nanoparticles (10–20 nm) and then coated with crystallized TiO2. These TiO2–graphene@Fe3O4 composites exhibited a high magnetic response to the external magnetic field. And the huge surface of the graphene dramatically boosted the adsorbability and charge mobility, which lowered the recombination rate of electron–hole pairs, and hence systematically enhanced photocatalytic activity. The combination of adsorption and photodegradation endowed the composite with a better performance in the removal of trace amounts of MC-LR than the commercial photocatalyst, Degussa P25. The concentration of MC-LR can be lowered to less than 1 μg L−1 (a provisional safety guideline by the World Health Organization) from 500 μg L−1 under UV light in 30 min. The loading of TiO2–graphene@Fe3O4, the pH, and the UV energy were also optimized. Moreover, the stable removal capability of TiO2–graphene@Fe3O4 was confirmed over multiple cycles. Finally, the removal performance was also evaluated under natural light illumination in real surface water samples. This work paves the way for the development of more efficient and easily separable purifiers for the removal of pollutants and toxins from contaminated water.

A simple, novel approach was developed for the preparation of boronic acid functionalized Fe3O4 magnetic nanoparticles (MNPs) via thiol–ene (TE) click reaction. In this work, two clickable Fe3O4 MNPs functionalized with either alkene or thiol moieties were synthesized. Firstly, Fe3O4 MNPs were synthesized through a solvothermal method and then the clickable alkene- or thiol-coated Fe3O4 MNPs were prepared by sol–gel reaction with an organosilicon coupling agent, 3-(methacryloyloxy) propyltrimethoxylsilane (MPS) or 3-mercaptopropyltriethoxysilane (MPTES). The carbon–carbon double bonds/thiol groups on the surface of the MNPs serve as clickable sites to react with 4-mercaptophenylboronic acid (4-MPBA)/3-acrylamidophenylboronic acid (AAPBA) during the subsequent TE click reaction. Finally, the high density of boronic acid ligands immobilized on the surface of the Fe3O4 MNPs was obtained via TE click reactions. The morphology, adsorption and recognition properties of the Fe3O4 MNPs were investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), vibrating sample magnetometry (VSM) and X-ray photoelectron spectrometry (XPS). Four proteins, including ovalbumin (OB) and transferrin (Trf) as glycoprotein templates, and lysozyme (Lyz) and horse heart cytochrome c (Cyt C) as non-glycoprotein templates are chosen as target proteins. Two types of click-Fe3O4 MNP (Fe3O4@MPS@PBA and Fe3O4@SH@AAPBA) exhibit a high binding capacity and excellent specificity towards glycoproteins, and can selectively capture and separate glycoproteins from egg white samples directly. Furthermore, this work could provide a promising method of surface modification for the design of more efficient adsorbents for the isolation and enrichment of proteins from complex bio-samples.

In this study, a facile and efficient separation of abundant proteins from bovine blood using core–shell structure nanoparticles with a magnetic core and an immobilized metal affinity ligand iminodiacetic acid (IDA) chelating Ni(II) is presented. Firstly, Fe3O4 magnetic nanoparticles (MNPs) were synthesized through a solvothermal method and then were conveniently surface-modified with 3-(methacryloyloxy) propyltrimethoxylsilane as anchor molecules to donate vinyl groups. Next a high density poly(4-vinylbenzylchloride) (PVBC) shell was synthesized on the surface of silica-coated Fe3O4 MNPs via distillation–precipitation polymerization. After the PVBC shell reacted with iminodiacetic acid (IDA) in alkaline aqueous solution, the magnetite was charged with Ni2+ to form Ni(II)-IDA functionalized hybrid Fe3O4@PVBC@IDA-Ni MNPs. Transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and a vibrating sample magnetometer (VSM) were employed to evaluate the size, morphology and magnetic property of the resulting magnetic nanospheres. The high saturation magnetization (48.1 emu g−1) provides the materials with the convenience of magnetic separation under an external magnetic field and they can be subsequently reused. The core–shell Fe3O4@PVBC@IDA-Ni MNPs exhibit excellent performance in the separation of protein bovine hemoglobin (BHb), and the binding capacity is as high as 1988 mg g−1. In addition, the Fe3O4@PVBC@IDA-Ni MNPs can be used in selective removal of abundant protein Hb in the bovine blood samples. This opens a novel route for its future application in removing abundant protein in proteomic analysis.

In this study, a boronate-silica hybrid affinity monolith was prepared for specific capture of glycoproteins at neutral pH condition. The monolith was synthesized via a facile one-pot procedure in a stainless steel column by concurrently mixing hydrolyzed alkoxysilanes tetramethoxysilane and vinyltrimethoxysilane, organic monomer 3-acrylamidophenylboronic acid and initiator 2,2′-azobisisobutyronitrile together. The polycondensation of alkoxysilanes and copolymerization of organic monomer and vinyl-silica monolith were carried out successively by reacting at different temperatures. After optimizing the preparation conditions, the resulting hybrid affinity monolith was systematically characterized and exhibited excellent affinity to both cis-diol-containing small molecules and glycoproteins at neutral and physiological pH, including adenosine, horseradish peroxidase, transferrin and ovalbumin. The binding capacity of ovalbumin on monolith was measured to be 2.5 mg g−1 at pH 7.0. Furthermore, the hybrid affinity monolith was applied to the separation of transferrin from bovine serum sample at a physiological condition. Good repeatability was obtained and the relative standard deviations of retention time were 1.15 and 4.77 % (n = 5) for run-to-run and column-to-column, respectively.

In this study, a novel approach was developed to synthesize aminophenylboronic acid functionalized magnetic nanoparticles (NPs) via Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click’ chemistry. Firstly, azide-functional Fe3O4 NPs were obtained by a two-step chemical modification process. Then, an alkyne-phenylboronic acid molecule was connected onto the surface of magnetite by the CuAAC reaction. The morphology, structure and composition of the synthesized nanocomposites were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), vibrating sample magnetometer (VSM), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectrometry (XPS). Five proteins, including ovalbumin (OB), transferrin (Trf), as glycoprotein templates and lysozyme (Lyz), bovine serum albumin (BSA), horse heart cytochrome c (Cyt C) as nonglycoprotein templates are chosen as target proteins. The as-prepared click-Fe3O4@APBA NPs with a mean diameter of 23.2 nm showed a strong magnetic response to an externally applied magnetic field and exhibited a high adsorption capacity and excellent specificity towards glycoproteins in comparison with nonglycoproteins. The click-Fe3O4@APBA NPs showed the higher adsorption capacity towards glycoproteins than the nonclick-Fe3O4@APBA NPs which were synthesized through a common nucleophilic substitution reaction. The greatly enhanced adsorption capacity towards glycoproteins demonstrated that the ‘click’ method presented great superiority in ligand immobilization. Finally, the click-Fe3O4@APBA NPs could efficiently enrich glycoproteins from real egg white samples as well.

In this study, we report a novel method to synthesize core–shell structured Fe3O4 nanoparticles (NPs) covalently functionalized with iminodiacetic acid (IDA) via click chemistry between the azide and alkyne groups and charged with Cu2+. Firstly, the Fe3O4@SiO2 NPs were obtained using tetraethoxysilane (TEOS) to form a silica shell on the surface of the Fe3O4 core. The azide group-modified Fe3O4@SiO2 NPs were obtained by a sol–gel process using 3-azidopropyltriethoxysilane (AzPTES) as the silane agent. Fe3O4@SiO2–N3 was directly reacted with N-propargyl iminodiacetic via click chemistry, in the presence of a Cu(I) catalyst, to acquire the IDA-modified Fe3O4 NPs. Finally, through the addition of Cu2+, the Fe3O4@SiO2–IDA-Cu NP product was obtained. The morphology, structure and composition of the NPs were characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The resulting NPs showed a strong magnetic response to an externally applied magnetic field, a high adsorption capacity and excellent specificity towards hemoglobin (Hb). In addition, the Fe3O4@SiO2–IDA-Cu NPs can be used for the selective removal of abundant Hb protein in bovine and human blood samples.

In this study, we report a facile method for the preparation of core–shell magnetic molecularly imprinted polymers (MIPs) for protein recognition. Uniform carboxyl group functionalized Fe3O4 nanoparticles (NPs) were synthesized using a solvothermal method. Magnetic MIPs were synthesized by self-polymerization of dopamine in the presence of template protein on the surface of the Fe3O4 NPs. A thin layer of polydopamine can be coated on Fe3O4 NPs via dopamine self-polymerization and the imprinted polydopamine shells can be controlled by the mass ratio of Fe3O4 NPs and dopamine. More importantly, there is a critical value of polydopamine shell thickness for the maximum rebinding capacity. The as-prepared lysozyme-imprinted Fe3O4@polydopamine NPs show high binding capacity and acceptable specific recognition behavior towards template proteins. This method provides the possibility for the separation and enrichment of abundant proteins in proteomic analysis.

In this study, we present a general method to prepare the core–shell magnetic molecularly imprinted polymers (MIPs) nanoparticles (NPs) for sulfamethazine (SMZ). The resulting Fe3O4@MIPs NPs possess a highly improved imprinting effect, fast adsorption kinetics and high adsorption capacity, and can be applied to extract sulfonamide in the poultry feed. In this protocol, the magnetite NPs were synthesized by co-precipitating Fe2+ and Fe3+ in an ammonia solution first. Silica was then coated on the Fe3O4 NPs using a sol–gel method to obtain silica shell magnetic NPs. Subsequently, the vinyl groups were grated onto silica-modified Fe3O4 surface by 3-methacryloyloxypropyltrimethoxysilane. Finally, the MIPs films were formed on the surface of Fe3O4@SiO2 by the copolymerization of vinyl end groups with functional monomer, methacrylic acid, cross-linking agent, ethylene glycol dimethacrylate, the initiator azo-bis-isobutyronitrile and template molecule, sulfamethazine. The morphology, magnetic, adsorption and recognition properties of Fe3O4@MIPs NPs were characterized using transmission electron microscope (TEM), scanning electron microscope (SEM), Fourier transform infrared (FT-IR) spectrometer, vibrating sample magnetometer (VSM) and re-binding experiments. The results showed that the binding sites of Fe3O4@MIPs were good accessibility, fast adsorption rate and the maximum adsorption capacity of Fe3O4@MIPs to SMZ was 344.8 μg g−1. The selectivity of the obtained Fe3O4@MIPs NPs were elucidated by the different rebinding capability of SMZ and structural related sulfonamides in the mixed solution. The results indicated that the Fe3O4@MIPs had high imprinting factor 9.5 and significant selectivity. A method was developed for enrichment and determination of SMZ in the poultry feed samples with recoveries of duck and chicken feed ranging from 63.3 to 76.5% and 68.7 to 74.7%, respectively and the relative standard deviations (RSD) (<6.7%).Highlights► We present a general method to prepare the core–shell magnetic MIPs nanoparticles (Fe3O4@MIPs) for sulfamethazine. ► The Fe3O4@MIPs possess a highly improved imprinting factor, fast adsorption kinetics and high adsorption capacity. ► The structure and affinity properties of the resulting Fe3O4@MIPs were characterized. ► We used Fe3O4@MIPs for enrichment and determination of SMZ in the poultry feed samples.

A general method to prepare thin, molecularly imprinted polymer (MIP) coatings on magnetic Fe3O4 nanoparticles (NPs) with a uniform core–shell structure for the recognition and enrichment of protein was developed. Four proteins (bovine serum albumin (BSA, pI = 4.9), bovine hemoglobin (BHb, pI = 6.9), bovine pancreas ribonuclease A (RNase A, pI = 9.4) and lysozyme (Lyz, pI = 11.2)) with different isoelectric points were chosen as the templates. The magnetic protein-MIPs were synthesized by combining surface imprinting and sol–gel techniques. The morphology, adsorption and recognition properties of the magnetic molecularly imprinted NPs were investigated by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR) spectroscopy and through the use of a vibrating sample magnetometer (VSM). In comparison with the use of Lyz, BSA and RNase A as template proteins, BHb-imprinted Fe3O4 showed the best imprinting effect and the highest adsorption capacity among the four proteins. The as-prepared Fe3O4@BHb-MIPs NPs with a mean diameter of 230 nm were coated with an MIP shell that was 10 nm thick, which enabled the Fe3O4@BHb-MIPs to easily reach adsorption equilibrium. A high magnetic saturation value of 25.47 emu g−1 for Fe3O4@BHb-MIPs NPs was obtained, which endowed the adsorbent with the convenience of magnetic separation under an external magnetic field. The resultant Fe3O4@BHb-MIPs NPs could not only selectively extract a target protein from mixed proteins but also specifically capture the protein BHb from a real sample of bovine blood. In addition, different batches of magnetic MIPs showed good reproducibility and reusability for at least six repeated cycles.

A macroporous boronate affinity monolithic column was prepared and applied to specifically capture glycoproteins using metal-organic gels (MOGs) as a porogenic template. This newly explored application of MOGs has proven to be a more convenient method for the formation of macropores in contrast to traditional porogenic methods. The poly (3-acrylamidophenylboronic acid-co-ethylene dimethacrylate) monolithic columns were synthesized in stainless columns by in situ polymerization. To fabricate the macroporous formation with a uniformed open-channel network, the preparation conditions, such as reaction temperature, the concentration of the MOGs and the ratio of monomers were systematically investigated. The prepared macroporous monoliths were characterized by scanning electron microscope (SEM) and mercury intrusion porosimetry. Furthermore, horseradish peroxidase (HRP) and transferrin (TF) were chosen as test glycoproteins, and the chromatographic analysis demonstrated that the macroporous boronate affinity monoliths exhibited a higher selectivity and better dynamic binding capacity toward glycoproteins compared with non-glycoproteins. The resulted affinity monolithic column was successfully employed to specifically capture TF from a bovine serum sample.Highlights► An alternative method was developed for the synthesis of a macroporous boronate affinity monolithic column in a convenient way using MOG as a porogenic template. ► The monolithic column had the advantage of good macropore distribution and permeability for further high-throughput and efficient separations of glycoproteins. ► A one-step purification of transferrin from bovine serum sample was of success.

Currently, small proteins imprinting are more reported since large proteins molecular imprinting faces challenge due to their bulk size and complex structure. In this work, bovine serum albumin (BSA) surface-imprinted magnetic polymer was successfully synthesized based on atomic transfer radical polymerization (ATRP) method in the presence of common monomer (N-isopropylacrylamide) with the assistant of basic functional monomer (N-[3-(dimethylamino)propyl]-methacrylamide), which provides a achievable attempt for imprinting larger target proteins based on the ATPR with the mild reaction conditions. The BSA-imprinted polymer exhibited higher adsorption capacity and selectivity to BSA over the non-imprinted polymer. Competitive adsorption tests indicated the BSA-imprinted polymer had better selective adsorption and recognition properties to BSA in the mixture. The obtained BSA-imprinted polymer was applied to bovine serum, which also showed selectivity to BSA. In addition, a conventional aqueous two-phase solution of PEG/sulphate was used as elution for adsorbed BSA, which was compared with common NaCl elution.

Both of the magnetic particle adsorption and aqueous two-phase extraction (ATPE) were simple, fast and low-cost method for protein separation. Selective proteins adsorption by carboxyl modified magnetic particles was investigated according to protein isoelectric point, solution pH and ionic strength. Aqueous two-phase system of PEG/sulphate exhibited selective separation and extraction for proteins before and after magnetic adsorption. The two combination ways, magnetic adsorption followed by ATPE and ATPE followed by magnetic adsorption, for the separation of proteins mixture of lysozyme, bovine serum albumin, trypsin, cytochrome C and myloglobin were discussed and compared. The way of magnetic adsorption followed by ATPE was also applied to human serum separation.

This paper reports the preparation of carbon nanotubes (CNTs) functionalized with molecularly imprinted polymers (MIPs) for advanced removal of estrone. CNTs@Est-MIPs nanocomposites with a well-defined core–shell structure were obtained using a semi-covalent imprinting strategy, which employed a thermally reversible covalent bond at the surface of silica-coated CNTs for a large-scale production. The morphology and structure of the products were characterised by transmission electron microscopy and Fourier transform infrared spectroscopy. The adsorption properties were demonstrated by equilibrium rebinding experiments and Scatchard analysis. The results demonstrate that the imprinted nanocomposites possess favourable selectivity, high capacity and fast kinetics for template molecule uptake, yielding an adsorption capacity of 113.5 μmol/g. The synthetic process is quite simple, and the different batches of synthesized CNTs@Est-MIPs nanocomposites showed good reproducibility in template binding. The feasibility of removing estrogenic compounds from environmental water using the CNTs@Est-MIPs nanocomposites was demonstrated using water samples spiked with estrone.

A new approach is reported on the use of poly(N-isopropylacrylamide) (PNIPAM)-coated molecularly imprinted beads (coated MIP beads) for controlling the release of protein. The coated MIP beads were composed of double layers, an internal thermosensitive lysozyme-imprinted layer, and an external PNIPAM layer. The coated MIP beads were prepared by two-step surface-initiated living-radical polymerization (SIP). In this systemic study, the coated MIP beads had good selectivity to the template protein (lysozyme) and temperature stimulus-responsive behavior, both of which were superior to those of MIP beads having a layer of thermosensitive lysozyme-imprinted polymer only. Using the coated MIP beads, reference proteins and the template lysozyme could be released separately at 38 °C and at 23 °C. The corresponding coated non-imprinted beads (coated NIP beads) did not have such double thermosensitive “gates” with specific selectivity for a particular protein. The proposed smart controlled imprinted system for protein is attractive for chemical carriers, drug-delivery system, and sensors.

Two types of macroporous organic polymer monoliths based on glycidyl methacrylate (GMA), 4-vinylbenzyl chloride (VBC) and divinylbenzene (DVB) were prepared inside stainless-steel tubes. Azide functionalities were firstly introduced on the surfaces of poly(GMA-co-DVB) and poly(VBC-co-DVB) monoliths to provide reactive sites for click chemistry. With the application of copper(I)-catalyzed (3 + 2) azide-alkyne cycloaddition, an in-column click-modification approach for covalent attachment of long alkyl chains onto polymer monoliths was developed. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by the scanning electron microscopy, mercury intrusion porosimeter, Fourier transform infrared spectroscopy, and elemental analyses, respectively. The chromatographic performances of the “clicked” stationary phases were demonstrated with the high separation efficiency for a variety of proteins within 4 min.

In this study, we report a simple method to coat mesoporous silica onto carbon nanotubes (CNTs) via a two-step procedure. Mesoporous CNTs@SiO2 composites have been obtained by extracting cetyltrimethylammonium bromide (CTAB) via an ion-exchange procedure after silica-coated carbon nanotubes were synthesized with the aid of the cationic surfactant CTAB. The coating process was explicitly investigated, and a possible formation mechanism of the mesoporous CNTs@SiO2 was proposed, which reveals that the ratio of CTAB/CNTs plays a critical role in the coating process. Furthermore, the pore size of the as-prepared mesoporous silica could be exactly controlled by using different amounts of the bromide surfactant CTAB. The obtained mesoporous CNTs@SiO2 composite nanomaterial was evaluated with three typical proteins, cytochrome c (Cyt c), bovine serum albumin (BSA) and lysozyme (Lyz), with different molecular sizes. The adsorption and desorption of binary mixtures of Cyt c and BSA, Cyt c and Lyz, and a ternary mixture of Cyt c, BSA and Lyz showed that the mesoporous CNTs@SiO2 are effective and highly selective adsorbents for Cyt c. The as-prepared mesoporous CNTs@SiO2 composites have shown effective performance in size-selective adsorption of biomacromolecules, demonstrating great potential in biomacromolecular separation.

In this study, we report a method to synthesize core–satellite structured Fe3O4/polydopamine/Au composite nanoparticles (NPs). Firstly, the Fe3O4/polydopamine composite NPs with a well-defined core–shell structure are obtained using dopamine self-polymerization to form thin, surface-adherent polydopamine films onto the surface of a Fe3O4 “core”. The polydopamine shell could be adjusted by controlling the experimental parameters such as reaction time and the reactant concentrations. Then, numerous “satellites” of gold nanoparticles were assembled on the surface of Fe3O4/polydopamine by reducing Au3+ between the Fe3O4/polydopamine solid and HAuCl4 solution. Next, 11-mercaptoundecanoic acid (11-MUA) forms a self-assembled monolayer of MUA on the surface of the Au NPs and polydopamine layer. Finally, IDA-Cu functionalized Fe3O4/polydopamine/Au composite NPs are obtained by the carboxyl groups of MUA reacting with iminodiacetic acid (IDA), charged with Cu2+. The IDA-Cu groups, acting as an “anchor”, are attached on the gold and the polydopamine surface is designed for capturing target molecules. The morphology, structure and composition of the nanocomposites are characterized by transmission electron microscopy (TEM), X-ray powder diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectrometry (XPS). The resulting Fe3O4/polydopamine/Au composite NPs show not only a strong magnetic response to an externally applied magnetic field, but are also highly specific to protein bovine hemoglobin (BHb), and removal of abundant protein BHb in the bovine blood as well. This opens a novel route for future application in removing abundant protein in proteomic analysis.

Synthetic core–shell molecularly imprinted polymers (MIPs) were prepared for the extraction of trace triclosan in environmental water samples. The synthesis process combined a surface molecular imprinting technique with a sol–gel process based on carbon nanotubes (CNTs) coated with silica. The morphology and structure of the products were characterized by transmission electron microscopy and Fourier transform infrared spectroscopy. The adsorption properties of the polymers were demonstrated by equilibrium rebinding experiments and Scatchard analysis. The prepared imprinted materials exhibited fast kinetics, high capacity and favorable selectivity. The process of synthesis was quite simple and different batches of MIPs and non-imprinted polymers (NIPs) showed good reproducibility in the template binding. The feasibility of determination of triclosan from real samples was testified using spiked river and lake water samples. The recoveries of river water and lake water samples were ranged from 92.1 to 95.3% and 90.7 to 93.6%, respectively, when the environmental water samples were spiked with 0.1, 0.3, and 0.5 μg L−1 of TCS. In addition, the reusability of MIPs and NIPs without any deterioration in capacity was demonstrated for at least 10 repeated cycles.

A simple one-step in situ “click” modification strategy was developed for the preparation of hydrophobic organic monolithic columns for the first time. The column morphology and surface chemistry of the fabricated monolithic columns were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, respectively. The chromatographic performances of the C8/C18 “click” monoliths were evaluated through the separation of a mixture of five proteins such as ribonuclease A, soybean trypsin inhibitor, cytochrome c, bovine haemoglobin and bovine serum albumin. Compared with the blank column, the higher hydrophobicity stationary phases obtained from the “clicked” modification have longer retention times and higher resolution for the five proteins. The separation of five proteins mixture on click C18 monolith with gradient elution at different flow rates was also investigated, the baseline separation of five proteins could be achieved at three different flow rates.

This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chromatographic strategies in the past ten years, covering the main research in these areas especially in China. The reviewed research focused on enzymatic micro-reactors and peptide separation in bottom-up approaches, and protein and peptide separation in top-down approaches. The new enzymatic micro-reactor is able to accelerate proteolytic reaction rate from conventionally a couple of hours to a few seconds, and the multiple dimensional chromatographic-separation with various models or arrays could sufficiently separate the proteomic mixture. These advances have significantly promoted the research of protein/peptide separation and identification in proteomics.

A thermosensitive macroporous hydrogel showing selectivity for the lysozyme was developed by an imprinting procedure that is based on metal coordinate interaction. A metal chelate monomer [N-(4-vinyl)-benzyl iminodiacetic acid] forming coordination complex with the template protein in the presence of Cu ions co-polymerized with N-isopropylacrylamide and acrylamide, using N,N-methylenebisacrylamide as the cross-linker to prepare the thermosensitive protein-imprinted hydrogel. The synergetic combination of the smart property of the macroporous thermosensitive hydrogel with the merits of the coordinate interaction improved the selectivity and adsorption capacity, with respect to template lysozyme. The macropores were created by the frozen polymerization, and the influences of frozen polymerization and the chelate monomer content on the hydrogel affinity were investigated. The imprinted hydrogel can respond not only to external stimuli, but also to the template protein with a certain degree of shrinking. In recognition of the protein, the interaction of the imprinted thermosensitive hydrogel to the protein can be switched between the coordinate effect and the electrostatic effect by adding or not adding Cu ions. Finally, this imprinted hydrogel was used to purify the template lysozyme from the mixture of proteins and the real sample, which demonstrated its high selectivity.

A new and facile fabricating method for lysozyme molecularly imprinted polymer beads (lysozyme-MIP beads) in aqueous media was presented. Mesoporous chloromethylated polystyrene beads (MCP beads) containing dithiocarbamate iniferter (initiator transfer agent terminator) were used as supports for the grafting of lysozyme imprinted copolymers with acrylamide and N,N′-methylenebisacrylamide through surface initiated living-radical polymerization (SIP). After the polymerization, a layer of lysozyme-MIP was formed on the MCP beads. The SIP allowed an efficient control of the grafting process and suppressed solution propagation. Therefore, the obtained lysozyme-MIP beads had a large quantity of well-distributed pores on the surface without any visible gel formation in solution and were more advantageous comparing with traditional MIPs which were prepared by traditionally initiated radical polymerization. The obtained composites were characterized by Fourier transform infrared spectroscopy, elemental analysis, nitrogen sorption analysis and scanning electron microscopy. Chromatographic behaviors of the column packed with lysozyme-MIP beads exhibited ability in separating lysozyme from competitive protein (bovine hemoglobin, bovine serum albumin, ovalbumin or cytochrome c) in aqueous mobile phase.

A novel solid phase extraction (SPE) method for determination of tetracyclines (TCs) in milk and honey samples by molecularly imprinted monolithic column was developed. Using tetracycline (TC) as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linker, methanol as the solvent, cyclohexanol and dodecanol as the mixed porogenic solvents, a TC imprinted monolithic column was prepared by in situ molecular imprinting technique for the first time, and the optimal synthesis conditions and the selectivity of TC imprinted monolithic column were investigated. The interfering substances in food samples and TCs can be separated successfully on imprinted column. Molecularly imprinted solid phase extraction (MISPE) coupling with C18 column was used to determinate the TCs in milk and honey. The recoveries of this method for six tetracyclines antibiotics such as tetracycline (TC), oxytetracycline (OTC), minocycline (MINO), chlortetracycline (CTC), metacycline (MTC) and doxycycline (DTC) were investigated, and high recoveries of 73.3–90.6% from milk samples and 62.6–82.3% from honey samples were obtained. A method for determination of TCs at low concentration level in milk and honey samples was successfully developed by using the monolithic column as the precolumn for solid phase extraction of six TCs compounds.

Molecularly imprinted polymers (MIPs), based on photografting surface-modified polystyrene beads as matrices, were prepared with acrylamide as the functional monomer, bovine hemoglobin as the template molecule and N, N′-methylene bisacrylamide as the crosslinker in a phosphate buffer. The results of IR, scanning electron microscope (SEM) and elemental analyses demonstrated the formation of a grafting polymer layer on the polystyrene-bead surface. Subsequent removal of the template left behind cavities on the surface of the polymer matrix with a shape and an arrangement of functional groups having complementary binding sites with the original template molecule. The adsorption studies showed that the imprinted polymers have a good adsorption capacity and specific recognition for bovine hemoglobin as the template molecule. Our results demonstrated that the polymer prepared via the photografting surface-modified method exhibited better selectivity for the template. Attempts to employ the new method in molecular imprinting techniques may introduce new applications for MIPs and facilitate probable protein separation and purification.

CdHgTe nanoparticles (NPs) with the emission in the near-infrared regions were prepared in aqueous solution, and were characterized by transmission electron microscopy, X-ray diffraction spectrometry, spectrofluorometry and ultraviolet-visible spectrometry. Based on the fluorescence quenching of CdHgTe NPs in the presence of proteins, a novel method for the determination of proteins with CdHgTe NPs as a near-infrared fluorescence probe was developed. Maximum fluorescence quenching was observed with the excitation and emission wavelengths of 500 and 693 nm, respectively. Under the optimal conditions, the calibration graphs were linear in the range of 0.04 × 10−6–5.6 × 10−6 g ml−1 for lysozyme (Lyz) and 0.06 × 10−6–6.1 × 10−6 g ml−1 for bovine hemoglobin (BHb), respectively. The limits of detection were 13 ng ml−1 for Lyz and 27 ng ml−1 for BHb, respectively. Four synthetic samples were determined and the results were satisfied.

Monolith with immobilized pH gradient (M-IPG) is a novel separation matrix for amphoteric substances, such as peptides and proteins. To improve the properties of the newly designed column, efforts were made to optimize the preparation procedure, including the concentrations of the monomers, glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA), as well as the kinds of porogens, which led to a monolith with improved permeability, uniformity and continuity. In addition, different diamines, including ethylenediamine, 1,3-diaminopropane, 1,4-diaminobutane and 1,5-diaminopentane, were subjected to aminate the polymer, and the last two exhibited excellent reactivity with epoxy on the polymer surface, which could obviously make the immobilization of pH gradient facilitated. Under the optimal conditions, a simple method to prepare M-IPG column with narrow pH gradient was developed with commercial carrier ampholytes (CAs) solution. Such columns were applied into the analysis of proteins and peptides, and showed the improved resolution compared to the traditional one with a wide pH distribution.

The influence of Joule heating on electroosmotic flow velocity, the retention factor of neutral analytes, and separation efficiency in capillary electrochromatography was investigated theoretically and experimentally. A plot of electrical current against the applied electrical field strength was used to evaluate the Joule heating effect. When the mobile phase concentration of Tris buffer exceeded 5.0 mM in the studied capillary electrochromatography systems using particulate and monolithic columns (with an accompanying power level of heat dissipation higher than 0.35 W/m), the Joule heating effect became clearly noticeable. Theoretical models for describing the variation of electroosmotic flow velocity with increasing applied field strength and the change of retention factors for neutral analytes with electrical field strength at higher Tris buffer concentrations were analyzed to explain consequences of Joule heating in capillary electrochromatography. Qualitative agreement between experimental data and implications of the theoretical model analysis was observed. The decrease of separation efficiency in capillary electrochromatography with macroporous octadecylsilica particles at high buffer concentration can be also attributed to Joule heating mainly via the increased axial diffusion of the analyte molecules and dispersion of solute bands by a nonuniform electroosmotic flow profile over the column cross-section. However, within a moderate temperature range, the contribution of the macroscopic velocity profile in the column arising from radial temperature gradients is insignificant.

A simple and sensitive method for the determination of short and long-chain fatty acids using high-performance liquid chromatography with fluorimetric detection has been developed. The fatty acids were derivatized to their corresponding esters with 9-(2-hydroxyethyl)-carbazole (HEC) in acetonitrile at 60°C with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride as a coupling agent in the presence of 4-dimethylaminopyridine (DMAP). A mixture of esters of C1–C20 fatty acids was completely separated within 38 min in conjunction with a gradient elution on a reversed-phase C18 column. The maximum fluorescence emission for the derivatized fatty acids is at 365 nm (λex 335 nm). Studies on derivatization conditions indicate that fatty acids react proceeded rapidly and smoothly with HEC in the presence of EDC and DMAP in acetonitrile to give the corresponding sensitively fluorescent derivatives. The application of this method to the analysis of long chain fatty acids in plasma is also investigated. The LC separation shows good selectivity and reproducibility for fatty acids derivatives. The R.S.D. (n=6) for each fatty acid derivative are <4%. The detection limits are at 45–68 fmol levels for C14–C20 fatty acids and even lower levels for