Copper-Catalyzed Difluoroalkylation Reaction.

This review describes recent advances in copper-catalyzed difluoroalkylation reactions. The RCF2 radical is generally proposed in the mechanism of these reactions. At present, various types of copper-catalyzed difluoroalkylation reactions have been realized. According to their characteristics, we classify these difluoroalkylation reactions into three types.

A CRISPR/Cas9-Gal4BD donor adapting system for enhancing homology-directed repair.

The fast-rising CRISPR-derived gene editing technologies has been widely used in the fields of life science and biomedicine, as well as plant and animal breeding. However, the efficiency of homology-directed repair (HDR), an important strategy for gene knock-in and base editing, remains to be improved. In this study, we came up with the term Donor Adapting System (DAS) to summarize those CRISPR/Cas9 systems modified with adaptor for driving aptamer-fused donor DNA. A set of CRISPR/Cas9-Gal4BD DAS was designed in our study. In this system, Gal4 DNA binding domain (Gal4BD) is used as adaptor to fuse with Cas9 protein, and Gal4 binding sequence (Gal4BS) is used as aptamer to bind to the double-stranded DNA (dsDNA) donor, in order to improve the HDR efficiency. Preliminary results from the HEK293T-HDR.GFP reporter cell line show that the HDR editing efficiency could be improved up to 2-4 times when donor homologous arms under certain length (100-60 bp). Further optimization results showed that the choice of fusion port and fusion linker would affect the expression and activity of Cas9, while the Cas9-Gal4BD fusion with a GGS5 linker was the prior choice. In addition, the HDR efficiency was likely dependent on the aptamer-dsDNA donor design, and single Gal4BD binding sequence (BS) addition to the 5'-end of intent dsDNA template was suggested. Finally, we achieved enhanced HDR editing on the endogenous AAVS1 and EMX1 sites by using the CRISPR/Gal4BD-Cas9 DAS, which we believe can be applied to facilitate animal molecular design breeding in the future.

Dual-function monolithic enzyme reactor based on dopamine/graphene oxide coating for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment.

A new dual-function enzyme reactor was prepared based on a dopamine/graphene oxide coated boron affinity monolithic column, which can be used for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment. Firstly, a boron affinity monolithic column was prepared as the carrier for enzyme reactor. Secondly, the monolithic column was coated with dopamine/graphene oxide to provide higher specific surface area for the increase in the amount of trypsin bound. Then, dopamine can self-polymerize under alkaline conditions to produce multiple reaction sites. By the Schiff base reaction and Michael addition reaction with amino, sulfhydryl groups to trypsin, enzyme were immobilized on the boron affinity monolithic carrier. The enzyme activity was characterized by kinetic parameters maximum rate (Vmax) of the enzyme reaction and Michaelis constant (Km). Km of the dual-function enzyme reactors doped with PDA/GO and without PDA/GO were 34.37 and 120.93 mM, Vmax were 1.35 and 3.35 mM/min, respectively. The performance of the dual-function enzyme reactor was evaluated by protein extraction of mouse liver. After digested by the dual-function enzyme reactor, the number of peptides was 4,801, which was 960 more than the number of peptides in the solution digestion. At the same time, the dual-function enzyme reactor displayed the ability to capture cis-dihydroxy compounds specificly. A total of 55 glycopeptides were enriched in the dual-functional enzyme reactor, corresponding to 33 glycoproteins. The dual-function enzyme reactor provided repeatable performance and robust with long-term storage.

Improving the Identification of Lysine-Acetylated Peptides Using a Molecularly Imprinted Monolith Prepared by a Deep Eutectic Solvent Monomer.

To overcome the identification challenge of low-abundance lysine acetylation (Kac), a novel approach based on a molecularly imprinted polymer (MIP) was developed to improve the extraction capacity of Kac peptides in real samples. Green deep eutectic solvents (DESs) were introduced and used as one of the synergistic functional monomers with zinc acrylate (ZnA). Glycine-glycine-alanine-lysine(ac)-arginine (GGAKacR) was chosen as a template and N,N'-methylenbisacrylamide (MBAA) was used as a cross-linker. The obtained GGAKacR-MIP had excellent selectivity for the template with an imprinting factor (IF) of up to 21.4. The histone digest addition experiment demonstrated that GGAKacR-MIP could successfully extract GGAKacR from a complex sample. Finally, the application to the extraction of Kac peptides from mouse liver protein digestion was studied in detail. The number of Kac peptides and Kac proteins identified was 130 and 110, which were 3.71-fold and 3.93-fold higher than those of the untreated sample. In addition, the number of peptides and proteins identified after treatment increased from 5535 and 1092 to 17 149 and 4037 (3.10-fold and 3.70-fold, respectively). The results showed that the obtained MIP may provide an effective technical tool for the identification of Kac-modification and peptide fractionation, as well as a potential approach for simultaneously identifying post-translational-modified proteomic and proteomic information.

Construction of a microfluidic platform integrating online protein fractionation, denaturation, digestion, and peptide enrichment.

Microfluidic system with multi-functional integration of high-throughput protein/peptide separation ability has great potential for improving the identification capacity of biological samples in proteomics. In this paper, a sample treatment platform was constructed by integrating reversed phase chromatography, immobilized enzyme reactor (IMER) and imprinted monolith through a microfluidic chip to achieve the online proteins fractionation, denaturation, digestion and peptides enrichment. We firstly synthesized a poly-allyl phenoxyacetate (AP) monolith and a lysine-glycine-glycine (KGG) imprinted monolith separately, and investigated in detail their performance in fractionating proteins and extracting KGG from the protein digests of MCF-7 cell. The removal percentage of 94.6% for MCF-7 cell protein and the recovery of 90.8% for KGG were obtained. The number of proteins and peptides identified on this microfluidic platform was 2,004 and 8,797, respectively, which was 2.8-fold and 3.0-fold higher than that of untreatment sample. The time consumed by this platform for a sample treatment was about 9.6 h, less than that of conventional method (approximate 13.3 h). In addition, this platform can enrich some peptide fragments containing KGG based on imprinted monolith, which can be served for the identification of ubiquitin-modified proteomics. The successful construction of this integrated microfluidic platform provides a considerable and efficient technical tool for simultaneous identification of proteomics and post-translational modification proteomics information.

Deep eutectic solvents-based polymer monolith incorporated with titanium dioxide nanotubes for specific recognition of proteins.

An organic-inorganic hybrid monolith incorporated with titanium dioxide nanotubes (TNTs) and hydrophilic deep eutectic solvents (DESs) was prepared and evaluated by the isolation of proteins using solid phase microextraction. A typical polymerization system was composed of choline chloride/methacrylic acid (ChCl/MAA, DESs monomer), glycidyl methacrylate (GMA), as well as ethylene glycol dimethacrylate (EDMA) in the presence of TNTs. Then the epoxy groups on the surface of the resulting monolith were modified with amino groups. The synergistic effect of TNTs and DESs monomer to improve the enrichment performance of the sorbent significantly was demonstrated. Compared with the corresponding TNTs/DESs-free monolith, the recoveries of BSA and OVA were increased to 98.6% and 92.7% (RSDs < 2.0%), with an improvement of more than 60.0%. With a correlation coefficient of determination (R2) higher than 0.9995, the enrichment factors (EFs) were 21.9-28.3-fold. In addition, the resulting monolith was further applied to specifically capture proteins from rat liver according to their pI value, followed by HPLC-MS/MS analysis. The results indicated that the developed monolith was an effective material to isolate protein species of interest according to the pI value of target proteins.

Preparation, characterization, and application of soluble liquid crystalline molecularly imprinted polymer in electrochemical sensor.

A novel soluble molecularly imprinted polymer (SMIP) without chemical cross-linker was successfully synthesized. The quinine (QN), which the structure was similar to the template, was chosen as the immobile template to improve the affinity of MIP. 4-Methyl phenyl dicyclohexyl ethylene (MPDE) was used as the liquid crystal (LC) monomer to increase the rigid of the composite. The cooperative effect of QN and MPDE was demonstrated by comparing with the conventional MIP, which synthesized without QN and MPDE. The polymerization conditions of SMIP including the ratio of MAA to MPDE, template to functional monomer, and HQN to QN were also optimized. Moreover, the characterizations of the SMIP were investigated by the transmission electron microscopy (TEM), field emission scanning electron microscopy (SEM), thermogravimetric analysis (TGA), X-ray diffraction (XRD), and nitrogen adsorption. In binding behavior, the SMIP presented the maximum adsorption capacity (0.37 ± 0.06 mmol/g) and imprinting factor (3.44 ± 0.25). And above all, the obtained polymer exhibited the solubility in the organic solution. In addition, the proposed SMIP as the electrochemical sensor exhibited a significant conductivity and sensitivity with the detection limit of 0.33 µM for HQN, the recoveries for the sample analysis varied from 97.4 to 100.8%, and the intra-day precision and inter-day precision were within 5.5% and 12.5%, respectively. It turned out that the SMIP had demonstrated more excellent potential than the traditional insoluble MIP in the development of the membrane-based electrochemical sensors.Graphical abstract.

Improving affinity of imprinted monolithic polymer prepared in deep eutectic solvent by metallic pivot.

One of the major drawbacks of conventional molecularly imprinted polymers (MIPs) is the requirements of volatility porogenic solvent during polymerization. To overcome the default, MIP based on deep eutectic solvent (DES, a new type of green designer solvents) has been synthesized successfully. To improve the affinity of the MIP based on DES, in this work, a strategy of metallic pivot was suggested in the first time to prepare a highly selective MIP monolithic column. A cetirizine-imprinted polymer was prepared in a DES-based porogen system composed of choline chloride/ ethylene glycol (ChCl-EG) in the presence of Co(Ac)2 as metallic pivot. The resulting DES- Co2+-MIP monolith had 23.5 times higher imprinting factor than the Co2+-free MIP monolith. The characterization of polymers indicated that DES was one of the primary factor influencing the MIP morphology and pore structure. Compared with previous metal-mediated and ionic liquid-based imprinted polymers, the introduction of DES as a porogen in polymerization led to higher imprinting factor (approximately 2.9 - 17.1 times). In addition, the resulting DES-Co2+-MIP can be used as an adsorbent for extraction of cetirizine from ethanol solution with the recoveries of 97.8%. As a conclusion, the metallic pivot is a rather valuable strategy for the synthesis of DES-based MIP monolith with high selectivity.

[Research progress in developing reporter systems for the enrichment of positive cells with targeted genome modification].

Targeted genome editing technology plays an important role in studies of gene function, gene therapy and transgenic breeding. Moreover, the efficiency of targeted genome editing is increased dramatically with the application of recently developed artificial nucleases such as ZFNs, TALENs and CRISPR/Cas9. However, obtaining positive cells with targeted genome modification is restricted to some extent by nucleases expression plasmid transfection efficiency, nucleases expression and activity, and repair efficiency after genome editing. Thus, the enrichment and screening of positive cells with targeted genome modification remains a problem that need to be solved. Surrogate reporter systems could be used to reflect the efficiency of nucleases indirectly and enrich genetically modified positive cells effectively, which may increase the efficiency of the enrichment and screening of positive cells with targeted genome modification. In this review, we mainly summarized principles and applications of reporter systems based on NHEJ and SSA repair mechanisms, which may provide references for related studies in future.

Current trends in the development of molecularly imprinted polymers in CEC.

This review focused on the developments in the field of molecularly imprinted polymers (MIPs) for CEC since 2009. New preparation techniques of MIP-based CEC, such as, portable microchip with macroporous monolithic imprinted microchannel, and low cross-linking MIPs based on liquid crystalline monomers, were discussed. Using selected cases rather than a comprehensive review of the entire field, our goal is to highlight the studies of the interest with an emphasis on recent work, and offers suggestions for future development in the field of imprinted materials for CEC separation.

Molecularly imprinted nanoparticles with nontailing peaks in capillary electrochromatography.

The combination of microparticles of molecularly imprinted polymers (MIPs) with partial filling capillary electrochromatography (CEC) has previously been demonstrated for the enantiomer separation. In this paper, precipitation polymerization was used to prepare d-zopiclone imprinted nanoparticles (50-80 nm) by a strategy of the dilution of pre-polymerization mixtures. The influence of some important parameters on the preparation of MIPs nanoparticles, including template to monomer ratio, type and amount of cross-linking monomer, and functional monomer composition ratio were investigated. In addition, the effect of separation condition, e.g., organic modifier content, pH value and salt concentration of buffer, on the electrochromatographic behavior of the MIP nanoparticles were studied. In spite of lower selectivity factor (1.11), high column performance (theoretical plates 41,400) of template was obtained and the resolution of enantiomers separation was 4.75 under the optimized conditions. Compared to the previously reported MIP microparticles, the MIP nanoparticles showed good peak symmetry and an ability of high speed separation (<15 min) in CEC mode.

Preparation and characterization of grafted imprinted monolith for capillary electrochromatography.

In this paper, a molecularly imprinted polymer (MIP) coating grafted to a trimethylolpropane trimethacrylate (TRIM) core material for CEC was reported. The core monolith was prepared with a solution of 20% (w/w) TRIM in a mixture of porogen and a polymerization precursor, which can generate a stable electroosmotic flow due to the formation of ionizable groups after postpolymerization hydrolization. Graft polymerization took place on the resultant TRIM monolith with a mixture of template, methacrylic acid, and ethylene glycol dimethacrylate. Strong recognition ability (selectivity factor was 5.83) for S-amlodipine and resolution of enatiomers separation (up to 7.99) were obtained on the resulting grafted imprinted monolith in CEC mode. The influence of CEC conditions on chiral separation, including the composition of mobile phase, pH value, and the operating voltages was studied. These results suggest that the method of grafted polymerization reported here allows a rapid development of MIP monolith once core materials with desired properties are available, and is a good alternative to prepare CEC-based monolithic MIPs.

Low crosslinking imprinted coatings based on liquid crystal for capillary electrochromatography.

Low loading capacity is the main problem of molecularly imprinted stationary phase, which is attributed to the high level of crosslinking restricting distortion phenomena of polymer backbone in molecular imprinting. A new approach based on liquid crystal with recognition ability is demonstrated for synthesis of molecularly imprinted polymer coatings in a low level of crosslinking. The resulting low crosslinking (20%) open-tubular imprinted capillary was able to separate enantiomers by means of capillary electrochromatography. The resolution of enantiomer separation achieved on the (S)-amlodipine-imprinted capillary was up to 6.36 in less than 2.5 min. The strong recognition ability with a selectivity factor of 1.81 and high column performance of template (up to 23,300 plates/m) were obtained. Performance of imprinting comparable to that recorded in conventional MIP stationary phase was observed. The liquid crystal MIP coatings were also prepared using either (S)-naproxen or (S)-ofloxacin as template molecule. The resolutions of enantiomers separation were 1.41 and 1.55, respectively. The results illustrate that the synthesis of low crosslinking MIP coatings based on liquid crystal is not only an experimental-simplified process of high performance, but also an approach to produce chiral stationary phase comparable to other chiral stationary phases.

Coatings of one monomer molecularly imprinted polymers for open tubular capillary electrochromatography.

One monomer molecularly imprinted polymer coatings were first synthesized in fused silica capillary columns with 2-methacrylamidopropyl methacrylate (MAM) as single functional monomer in addition to a cross-linking monomer. Since MAM may generate no or little EOF, a strategy of precursor of polymerization, which does not interfere with the formation of defined imprints, was used to introduce an ionizable functional monomer to generate a stable electroosmotic flow for electrochromatography (CEC) by post-polymerization hydrolization. The resulting MAM-based open-tubular imprinted capillary was able to separate enantiomers by means of CEC. The resolution of enantiomers separation achieved on S-amlodipine-imprinted capillary was up to 16.1. The strong recognition ability (selectivity factor was 3.23) and high column performance (theory plates was 26,053 plates m(-1)) of template were obtained. The MIP coatings were also prepared using either S-naproxen or S-ketoprofen as template molecule. The resolutions of enantiomers separation were 2.20 and 4.56, respectively. The results illustrate that the synthesis of MIP using one monomer is not only an experimental-simplified process, but also an approach to producing chiral stationary phase with high efficiency and selectivity.