Synthesis of micron-sized magnetic agarose beads chelated with nickel ions towards the affinity-based separation of histidine-tagged/rich proteins.

Developing high-performance magnetic particles for the effective separation and purification of target proteins has become an important topic in the area of biomedical research. In this work, a simple and novel strategy was proposed for fabricating magnetic Fe3O4@agarose-iminodiacetic acid-Ni microspheres (MAIN), which can efficiently and selectively isolate histidine-tagged/rich proteins (His-proteins). Based on the thermoreversible sol-gel transition of agarose, basic magnetic agarose microspheres were prepared through the inverse emulsion method, in which the emulsion contained agarose and amine-modified Fe3O4 nanoparticles. The size of the emulsion was controlled by the emulsification of a high-speed shear machine, which improved the specific surface area of MAIN. Subsequently, the amine-modified Fe3O4 nanoparticles were covalently crosslinked with agarose through epichlorohydrin, which could avoid leakage of the magnetic source during use and increase the stability of MAIN. The microsized MAIN exhibited a clearly visible spherical core-shell structure with a diameter range from 3.4 µm to 9.8 µm, and excellent suspension ability in aqueous solution. The maximum adsorption capacity of MAIN for histidine-rich bovine hemoglobin was 1069.2 mg g-1 at 35 °C, which was higher than those of commercialized and most reported magnetic agarose microspheres/nanoparticles. The MAIN showed excellent adsorption ability and selectivity toward His-proteins in a mixture of histidine-rich bovine serum albumin (BSA) and histidine-poor lysozyme (LYZ). When the amount of LYZ was 5-fold higher than that of BSA, the recovery of BSA reached 75.0%. To prove its practicability, MAIN was successfully employed for the enrichment of histidine-tagged RSV-F0 from the cell culture medium supernatant. According to the optimized conditions, MAIN could enrich approximately 0.1 mg of RSV-F0 from 1 mL of complex biological sample. Therefore, we believe that the novel MAIN could be applicable for efficient separation and purification of His-proteins from complex biological systems.

Modeling Viscoelasticity and Dynamic Nematic Order of Exchangeable Liquid Crystal Elastomers.

Exchangeable liquid crystal elastomers (XLCEs), an emerging class of recyclable polymer materials, consist of liquid crystalline polymers which are dynamically crosslinked. We develop a macroscopic continuum model by incorporating the microscopic dynamic features of the cross-links, which can be utilized to understand the viscoelasticity of the materials together with the dynamic nematic order. As applications of the model, we study the rheological responses of XLCEs in three cases: stress relaxation, strain ramp, and creep compliance, where the materials show interesting rheology as an interplay between the dynamic nematic order of the mesogenic units, the elasticity from the network structure, and the dissipation due to chain exchange reactions. Not only being useful in understanding the physical mechanism underlying the fascinating characteristics of XLCEs, this work can also guide their future fabrications with desired rheological properties.

Bioconjugation of nanozyme and natural enzyme to enable a one-step cascade reaction for the detection of metabolites.

Nanozyme, with enzyme-mimicking activity and excellent stability, has attracted extensive attention. However, some inherent disadvantages, including poor dispersion, low selectivity, and insufficient peroxidase-like activity, still limit its further development. Therefore, an innovative bioconjugation of a nanozyme and natural enzyme was conducted. In the presence of graphene oxide (GO), histidine magnetic nanoparticles (H-Fe3O4) were first synthesized by a solvothermal method. The GO-supported H-Fe3O4 (GO@H-Fe3O4) exhibited superior dispersity and biocompatibility because GO was the carrier and possessed outstanding peroxidase-like activity because of the introduction of histidine. Furthermore, the mechanism of the peroxidase-like activity of GO@H-Fe3O4 was the generation of •OH. Uric acid oxidase (UAO) was selected as the model natural enzyme and covalently linked to GO@H-Fe3O4 with hydrophilic poly(ethylene glycol) as a linker. UAO could specifically catalyze the oxidation of uric acid (UA) to generate H2O2, and subsequently, the newly produced H2O2 oxidized the colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue ox-TMB under the catalysis of GO@H-Fe3O4. Based on the above cascade reaction, the GO@H-Fe3O4-linked UAO (GHFU) and GO@H-Fe3O4-linked ChOx (GHFC) were used for the detection of UA in serum samples and cholesterol (CS) in milk, respectively. The method based on GHFU exhibited a wide detection range (5-800 µM) and a low detection limit (1.5 µM) for UA, and the method based on GHFC exhibited a wide detection range (4-400 µM) and a low detection limit (1.13 µM) for CS. These results demonstrated that the proposed strategy had great potential in the field of clinical detection and food safety.

Linking peptide-oriented surface imprinting magnetic nanoparticle with carbon nanotube-based fluorescence signal output device for ultrasensitive detection of glycoprotein.

Determination of trace glycoprotein has important guiding significance in clinical diagnosis and is usually achieved by immunoaffinity. However, immunoaffinity possesses inherent drawbacks, such as poor probability of high-quality antibodies, instability of biological reagents, and harmfulness of chemical labels to the body. Herein, we propose an innovative method of peptide-oriented surface imprinting to fabricate artificial antibody for recognition of glycoprotein. By integrating peptide-oriented surface imprinting and PEGylation, an innovative hydrophilic peptide-oriented surface imprinting magnetic nanoparticle (HPIMN) was successfully fabricated with human epidermal growth factor receptor-2 (HER2) as a model glycoprotein template. In addition, we further prepared a novel boronic acid-modified/fluorescein isothiocyanate-loaded/polyethylene glycol-covered carbon nanotube (BFPCN) as fluorescence signal output device, which was loaded with numerous fluorescent molecules could specifically label the cis-diol of glycoprotein at physiological pH via boronate-affinity interaction. To prove the practicability, we proposed a HPIMN-BFPCN strategy, in which the HPIMN first selectively captured the HER2 due to the molecular imprinted recognition and then the BFPCN specific labeled the exposed cis-diol of HER2 based on the boronate-affinity reaction. The HPIMN-BFPCN strategy exhibited ultrahigh sensitivity with limit of detection of 14 fg mL-1 and was successfully used in the determination of HER2 in spiked sample with recovery and relative standard deviation in the range of 99.0%-103.0% and 3.1%-5.6%, respectively. Therefore, we believe that the novel peptide-oriented surface imprinting has great potential to become an universal strategy for fabrication of recognition units for other protein biomarkers, and the synergy sandwich assay could become a powerful tool in prognosis evaluation and clinical diagnosis of glycoprotein-related diseases.