Magnetism-Enhanced Monolith-Based In-Tube Solid Phase Microextraction

Monolith-based in-tube solid phase microextraction (MB/IT-SPME) has received wide attention because of miniaturization, automation, expected loading capacity, and environmental friendliness. However, the unsatisfactory extraction efficiency becomes the main disadvantage of MB/IT-SPME. To overcome this circumstance, magnetism-enhanced MB/IT-SPME (ME-MB/IT-SPME) was developed in the present work, taking advantage of magnetic microfluidic principles. First, modified Fe₃O₄ nanoparticles were mixed with polymerization solution and in situ polymerized in the capillary to obtain a magnetic monolith extraction phase. After that, the monolithic capillary column was placed inside a magnetic coil that allowed the exertion of a variable magnetic field. The effects of intensity of magnetic field, adsorption and desorption flow rate, volume of sample, and desorption solvent on the performance of ME-MB/IT-SPME were investigated in detail. The analysis of six steroid hormones in water samples by the combination of ME-MB/IT-SPME with high-performance liquid chromatography with diode array detection was selected as a paradigm for the practical evaluation of ME-MB/IT-SPME. The application of a controlled magnetic field resulted in an obvious increase of extraction efficiencies of the target analytes between 70% and 100%. The present work demonstrated that application of different magnetic forces in adsorption and desorption steps can effectively enhance extraction efficiency of MB/IT-SPME systems

Flow cytometry assay for InaK-Ns and InaK-N/ARG1s.

<p>Cells containing different vectors labeled with Ddylight649-conjugated antibody against the HA epitope tag were analyzed by flow cytometry. The excitation laser was 638nm, and the emission filter was 660/20 BP. A-M indicated cells containing the pET23a-T empty vector; pET23a-InaK-N vector; pET23a <i>-</i>ssMalE-InaK-N vector; pET23a-ssTorA-InaK-N vector; pET23a-D₆-InaK-N vector; pET23a-E₆-InaK-N vector; pET23a-K₆-InaK-N vector; pET23a-InaK-N/ARG1 vector; pET23a-ssMalE-InaK-N/ARG vector; pET23a-ssTorA-InaK-N/ARG1 vector; pET23a-D₆-InaK-N/ARG1 vector; pET23a-E₆-InaK-N/ARG1 vector; and pET23a-K₆-InaK-N/ARG1 vector, respectively.</p

Surface Immobilization of Human Arginase-1 with an Engineered Ice Nucleation Protein Display System in <i>E</i>. <i>coli</i>

<div><p>Ice nucleation protein (INP) is frequently used as a surface anchor for protein display in gram-negative bacteria. Here, MalE and TorA signal peptides, and three charged polypeptides, 6×Lys, 6×Glu and 6×Asp, were anchored to the N-terminus of truncated INP (InaK-N) to improve its surface display efficiency for human Arginase1 (ARG1). Our results indicated that the TorA signal peptide increased the surface translocation of non-protein fused InaK-N and human ARG1 fused InaK-N (InaK-N/ARG1) by 80.7% and 122.4%, respectively. Comparably, the MalE signal peptide decreased the display efficiencies of both the non-protein fused InaK-N and InaK-N/ARG1. Our results also suggested that the 6×Lys polypeptide significantly increased the surface display efficiency of K₆-InaK-N/ARG1 by almost 2-fold, while also practically abolishing the surface translocation of non-protein fused InaK-N, indicating the interesting roles of charged polypeptides in bacteria surface display systems. Cell surface-immobilized K₆-InaK-N/ARG1 presented an arginase activity of 10.7 U/OD₆₀₀ under the optimized conditions of 40°C, pH 10.0 and 1 mM Mn²⁺, which could convert more than 95% of L-Arginine (L-Arg) to L-Ornithine (L-Orn) in 16 hours. The engineered InaK-Ns expanded the INP surface display system, which aided in the surface immobilization of human ARG1 in <i>E</i>. <i>coli</i> cells.</p></div

Fluorescence microscope assay of InaK-N/ARG1s.

<p>Surface fluorescence of the cells harboring various InaK-N/ARG1s under a fluorescence microscope with the excitation laser of 488nm, and the EGFP detection channel being used. A-G indicated cells containing empty pET23a-T vector; pET23a-InaK-N/ARG1 vector; pET23a-ssMalE-InaK-N/ARG1 vector; pET23a-ssTorA-InaK-N/ARG1 vector; pET23a-D₆-InaK-N/ARG1 vector; pET23a-E₆-InaK-N/ARG1 vector; and pET23a-K₆-InaK-N/ARG1 vector, respectively. 1: detecting of FITC signal, 2: the bright field, 3: the merge of FITC signal and bright field.</p

The chemical reagents used in this study.

<p>The chemical reagents used in this study.</p

Characterization of the cell surface-immobilized InaK-N-E/ARG1s.

<p>(A) Relative activity of the cells bearing K₆-InaK-N/ARG1 at different pH values. (B) Relative activity of the cells bearing K₆-InaK-N/ARG1 at different temperatures. (C) Operational stability of the cells bearing K₆-InaK-N/ARG1. The maximum activity was normalized to 100% in all of the assays. (D) MC trace analysis of the hydrolysis of molecular standard L-Orn. (E) MC trace analysis of the hydrolysis of molecular standard L-Arg.(F) MC trace analysis of the hydrolysis of L-Arg catalyzed by the cells bearing K₆-InaK-N/ARG1.</p

Sequences of the export signals and polypeptides used in this study.

<p>Sequences of the export signals and polypeptides used in this study.</p

DataSheet_1_Phenotypic variability and genetic diversity analysis of cultivated potatoes in China.docx

Phenotypic evaluation and molecular biotechnology are both important in the identification and utilization of crop germplasm resources. In this study, the phenotypic variation and genetic diversity of 149 main potato cultivars in China were investigated with 12 phenotypic traits and 24 SSR markers. The coefficient of variation of 12 phenotypic traits ranged from 12.11% to 156.93%. The results of SSR markers exhibited a relatively high level of genetic variation (Na =5.458 ± 1.499, Ne =3.300 ± 1.087, I =1.397 ± 0.298, Ho =0.797 ± 0.178, He = 0.660 ± 0.117, and PIC=0.702 ± 0.087). Population structure and phylogenetic tree analysis divided the varieties into three subgroups. The results indicated that ninety percent of the molecular variance was attributed to within-group differences, and the remaining 10% was attributed to variation among groups. Consistent with previous report, alleles of the STI032 marker were significantly associated with tuber starch content and growth period traits in the population. The results of this study could facilitate the utilization of potato germplasm resources, molecular genetic breeding and improvement.</p

Constructs in the study.

<p>(A) Different N-terminal fusions of InaK-N. (B) Different InaK-Ns fused with C-terminal human ARG1. (C) Schematic diagram of the cloning method for making the constructs. (D) <i>InaK-Ns</i> PCR products. Lanes 1–6 are PCR products for <i>InaK-N</i>, <i>ssMalE</i>-<i>InaK-N</i>, <i>ssTorA</i>-<i>InaK-N</i>, <i>D</i>_<i>6</i>-<i>InaK-N</i>, <i>E</i>_<i>6</i>-<i>InaK-N</i>, and <i>K</i>_<i>6</i>-<i>InaK-N</i>. (E) PCR products for <i>InaK-N/ARG1s</i>. Lane 1–8 <i>InaK-N</i>, <i>human ARG1</i>, <i>InaK-N/ARG1</i>, <i>ssMalE</i>-<i>InaK/ARG1</i>, <i>ssTorA</i>-<i>InaK-N/ARG1</i>, <i>D</i>_<i>6</i>-<i>InaK-N/ARG1</i>, <i>E</i>_<i>6</i>-<i>InaK-N/ARG1</i>, and <i>K</i>_<i>6</i>-<i>InaK-N/ARG1</i>. M: DNA Ladder.</p

Immobilization of the ARG1 fusions to the cell outer membrane.

<p>(A) SDS-PAGE and Western-Blot analysis of the human ARG1 fusions on the outer membrane. Upper panel is the SDS-PAGE analysis and the lower panel is the western-blot analysis. M: protein ladder; T: total cell proteins; OM: outer membrane proteins. InaK-N/ARG1; ssTorA-InaK-N/ARG1; K₆-InaK-N/ARG1; E₆-InaK-N/ARG1; D₆-InaK-N/ARG1; and ssMalE-InaK-N/ARG1 presented the recombinant strain expressing these fusion proteins, respectively; Control presented the recombinant strain containing empty pET23a-T. (B) Enzyme activity and proteinase K accessibility of the InaK-N/ARG1s. Proteinase K-: recombinant strain was untreated with proteinase K; Proteinase K+: recombinant strain was treated with proteinase K. K6-InaK-N/ARG1; E6-InaK-N/ARG1; D6-InaK-N/ARG1; ssMalE-InaK-N/ARG1; K6-InaK-N/ARG1 presented the recombinant strains expressing these fusion proteins, respectively.</p