Automated high performance liquid chromatography with on-line reduction of disulfides and chemiluminescence detection for determination of thiols and disulfides in biological fluids

In general, the reduction of disulfide bonds with tris(2-carboxyethyl)phosphine (TCEP) is performed using off-line operation, which is not only time-consuming but also vulnerable to the spontaneous re-oxidation of thiols during sample preparation and subsequent analysis procedures. To the best of our knowledge, there has been not any case on the on-line reduction for biological disulfides coupled with high performance liquid chromatography (HPLC). In this study, these obstacles are overcome by packing Zn(II)-TCEP complexes into a home-made column. The as-synthesized Zn(II)-TCEP complexes enable efficient reduction of disulfide bonds at pH 3.0. This acidic pH value was compatible with that of the mobile phase for HPLC separation of thiols and disulfides. Therefore, using fluorosurfactant-prepared triangular gold nanoparticles as HPLC postcolumn specific chemiluminescence (CL) reagents for thiols, the feasibility of the established on-line reduction column has been confirmed for the direct identification of both thiols and disulfides by incorporating this reduction column into a single chromatographic separation. Detection limits for these analytes range from 8.3 to 25.4 nM and the linear range in a log–log plot can comprise three orders of magnitude. Finally, the utility of this automated on-line reduction of disulfides-HPLC-CL system has been demonstrated for the reliable determination of thiols and disulfides in human urine and plasma samples.     

Sensitive chemiluminescence aptasensor based on exonuclease-assisted recycling amplification

We report herein an exonuclease-assisted aptamer-based target recycling amplification strategy for sensitive and selective chemiluminescence (CL) determination of adenosine. This aptasensor is based on target-induced release of aptamers from capture probes immobilized on the 96-well plate surface, and thus leading to a decreased hybridization with gold nanoparticle-functionalized reporter sequences followed by a CL signal. The introduction of exonuclease III catalyzes the stepwise removal of mononucleotides from 3′-hydroxyl termini of duplex DNAs of aptamers, liberating the adenosine. Therefore, a single copy of target adenosine can lead to the release and digestion of numerous aptamer strands from the 96-well plates and ultimately an enhanced sensitivity is achieved. Experimental results revealed that the exonuclease-assisted recycling strategy enabled the monitoring of adenosine with wide working ranges and low detection limits (LOD: 0.5 nM). This new CL strategy might create a novel technology for the detection of various targets and could find wide applications in the environmental and biomedical fields.     

Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.     

Droplet-based microfluidics for dose–response assay of enzyme inhibitors by electrochemical method

A simple but robust droplet-based microfluidic system was developed for dose–response enzyme inhibition assay by combining concentration gradient generation method with electrochemical detection method. A slotted-vials array and a tapered tip capillary were used for reagents introduction and concentration gradient generation, and a polydimethylsiloxane (PDMS) microfluidic chip integrated with microelectrodes was used for droplet generation and electrochemical detection. Effects of oil flow rate and surfactant on electrochemical sensing were investigated. This system was validated by measuring dose–response curves of three types of acetylcholinesterase (AChE) inhibitors, including carbamate pesticide, organophosphorus pesticide, and therapeutic drugs regulating Alzheimer's disease. Carbaryl, chlorpyrifos, and tacrine were used as model analytes, respectively, and their IC₅₀ (half maximal inhibitory concentration) values were determined. A whole enzyme inhibition assay was completed in 6 min, and the total consumption of reagents was less than 5 μL. This microfluidic system is applicable to many biochemical reactions, such as drug screening and kinetic studies, as long as one of the reactants or products is electrochemically active.     

(Δ)[Fe(II)(phen)3]2+ Filled in the Chiral Cavitiy Constituted by {(Δ)[Fe(III)(C2O4)3](NH42+)(DMF)(H2O)32−}n

A chiral coordination compound {(Δ)[Fe(II)(phen)₃][(Δ)Fe(III)(C₂O₄)₃](NH₄)(H₂O)₃(DMF)}_n (phen = 1,10‐phenanthroline), (DMF = N,N'‐Dimethylformamide), has been synthesized, and the structure has been revealed by infrared spectroscopy and X‐ray single‐crystal diffraction. The framework consists of two chiral subunits. One subunit (Δ)[(Fe(III)(C₂O₄)₃]^3? which as host anion forms a chiral porous three‐dimensional supermolecular network with lattice water, lattice DMF and lattice ammonium cation through hydrogen bonds. And then the other is Δ[Fe(II)(phen)₃]²⁺ which as guest cation fills in the chiral cavity located in the previously mentioned host porous network.     

Effects of Divalent Metal Ions on the Chaperone Activity and Structure of Rat Lens H18G Mutant αB‐Crystallin

αB‐crystalin, a small heat shock protein and a component of α‐crystalin, is a molecular chaperone playing an important role in preventing the formation of cataracts. It has been reported that His18 is an important site for Cu²⁺ to bind with to form a stable metal complex and thus to enhance this chaperone‐like activity of human αB‐crystalin. In this work, we used site‐directed mutagenesis to clone and express H18G rat lens αB‐crystalin in order to investigate the role of His18 in chaperoning activity. We found that 1 mM of Cu²⁺, or Zn²⁺, rather than Mg²⁺, significantly enhanced the chaperone‐like activity of wild type αB‐crystalin. Whereas, it is Zn²⁺ and Mg²⁺, not Cu²⁺, that significantly reduced this activity of H18G αB‐crystalin. In the absence of cation, H18G showed better activity compared to the wild type αB‐crystalin. ANS fluorescence measurement showed there was no linear relationship between chaperone‐like activity and surface hydrophobicity, indicating that surface hydrophobicity is not a prerequisite for chaperone‐like activity. An HPLC size‐exclusion chromatography study showed that in the presence of metal ions, wild type αB‐crystalin tended to aggregate via dissociation and re‐association into a high molecular aggregate with a molecular weight higher than 1400 kDa and then precipitated, suggesting that the presence of metal ions is a factor leading to the formation of cataracts. Both the near and far UV‐CD spectra suggested that the wild type αB‐crystalin reflected more β‐sheet structural characteristics; whereas the H18G reflected more random coil characteristics. The H18G induced structural alterations as to develop more random coil characteristics and more micro‐environmental changes around the tryptophan residues. This work suggested that His18 may not be a crucial binding site for Cu²⁺, but rather that it may be an important binding site for Zn²⁺ in terms of chaperone‐like activity and the process of metal induced self‐aggregation is prerequisite for chaperone‐like activity to occur.     

Experimental Evidence for Ion Accumulation Time Affecting Qualitative and Quantitative Analysis of Ophiopogons in Ophiopogon Extract by Hybrid Ion Trap Time-of-Flight Mass Spectrometry

The qualitative and quantitative capability of the ion trap mass analyzer could be greatly affected by the accumulation time. However, the importance of the accumulation time has not so far been thoroughly explored. Here, the influence of ion accumulation time on qualitative and quantitative analysis of complicated components was systematically investigated based on the case study of 40 ophiopogonins in Ophiopogon extract by hybrid ion trap time-of-flight mass spectrometry (LCMS-IT-TOF). In this process, the accumulation time was set at 10, 25, 50, 100, and 200 ms, respectively. The effect of accumulation time on qualitative analysis of ophiopogonins was studied by comparing the total ion current (TIC) of MS¹, TIC of MS², and the number and signal of fragmental ions. The results demonstrated that the signal could be greatly influenced by varying the accumulation time. The number and signal of the fragmental ions were increased significantly with a longer accumulation time in the range of 10–100 ms. Also, the effect of accumulation time on quantitative analysis of ophiopogonins was investigated by comparing the linearity, accuracy, and precision measured on LCMS-IT-TOF. Importantly, quantitative parameters could all be significantly improved by choosing an appropriate accumulation time.

     

The molecular diversity of three-component reactions of 4-dimethylamino- or 4-methoxypyridine with acetylenedicarboxylates and arylidene cyanoacetates

The three-component reactions of 4-dimethylamino- or 4-methoxypyridine with acetylenedicarboxylates and arylidene cyanoacetates showed very interesting molecular diversity. The polysubstituted 1,8,9,9a-tetrahydro-4H-1,4-ethanoquinolizines, 2H-pyran-2,3-dicarboxylates, and buta-1,3-diene-1,2,4-tricarboxylates derivatives can be formed in high yields and with good diastereoselectivity depending on the substrates and reaction conditions.     

Macrocycles consisting of flexible and rigid segments: enforced folding and host-guest inclusion exciplex formation

Macrocycles consisting of two tris(phenylene ethynylene) (or tri-PE) units connected via two flexible linkers adopt an ‘unfolded’ conformation that is converted into a folded conformation upon introducing intramolecular hydrogen bonding interaction. These foldable macrocycles are capable of forming inclusion charge transfer (CT) complex with electron-deficient small aromatics.     

Palladium(II)-catalyzed tandem annulation reaction of o-alkynylbenzoates with methyl vinyl ketone for the synthesis of isocoumarins

A palladium(II)-catalyzed highly regioselective tandem reactions of o-alkynylbenzoates with methyl vinyl ketone for the synthesis of isocoumarins was developed. It is a convenient, mild and environmentally benign reaction with moderate to high yield. The reaction is initiated by the Pd(II) species and regenerate the Pd(II) species to complete the catalytic cycle without the necessity of a redox system.