Double-layer poly(vinyl alcohol)-coated capillary for highly sensitive and stable capillary electrophoresis and capillary electrophoresis with mass spectrometry glycan analysis.

Glycosylation plays an important role in protein conformations and functions as well as many biological activities. Capillary electrophoresis combined with various detection methods provided remarkable developments for high-sensitivity glycan profiling. The coating of the capillary is needed for highly polar molecules from complex biosamples. A poly(vinyl alcohol)-coated capillary is commonly utilized in the capillary electrophoresis separation of saccharides sample due to the high-hydrophilicity properties. A modified facile coating workflow was carried out to acquire a novel multiple-layer poly(vinyl alcohol)-coated capillary for highly sensitive and stable analysis of glycans. The migration time fluctuation was used as index in the optimization of layers and a double layer was finally chosen, considering both the effects and simplicity in fabrication. With migration time relative standard deviation less than 1% and theoretical plates kept stable during 100 consecutive separations, the method was presented to be suitable for the analysis of glycosylation with wide linear dynamic range and good reproducibility. The glycan profiling of enzymatically released N-glycans from human serum was obtained by the presented capillary electrophoresis method combined with mass spectrometry detection with acceptable results.

From macro- to nano- scales: Effect of fibrillary celluloses from okara on performance of edible starch film.

Starch/cellulose composite is one of the most promising systems since both matrix and reinforce agent have same chemical unite glucose, which results in an excellent compatibility. In this work, edible starch film was developed by compositing starch with diverse fibrillary celluloses (FCs) derived from okara, employing a confluence of chemical interactions and mechanical influences. Since diameter of the FCs can be easily controlled by processing methodologies, it is the first time to systematically investigate the effect of diameter of the FCs from macro to nano-scales on the performances of starch-based film. The fabricated macro- and nano-fibrillar celluloses and reinforced starch films were characterized by scanning electron microscope, optical microscopy, Fourier transform infrared spectroscopy, Rheometer and contact angle. Results showed that the FCs increased modulus (about 170 %) and tensile strength (about 180 %) significantly as expected since they are well-compatible and some chemical interactions. It was found that nano-fibrillary celluloses (CNFs) improve the toughness (about 20 %) of the starch film more efficiently, which improved the well-recognized weakness of starch-based materials. The nano-scale roughness on the surface of the starch film caused by different shrinkage ratios between starch and CNFs during drying reduced water sensitivity, which is another well-recognized weakness of starch film.

The coating of smart pH-responsive polyelectrolyte brushes in capillary and its application in CE.

A novel pH-responsive coating technique was developed and applied to CE successfully in this paper. The coating was formed by bonding mixed opposite charge poly(acrylic acid) and poly(2-vinylpyridine) randomly onto the inner wall of a silica capillary. The coating processes were first characterized by ellipsometry and atomic force microscopy at macroscale and microscale, respectively. Measurements of EOF were implemented to confirm the coating. Direction and velocity of EOF became controllable from negative to positive, showing a perfect sigmoidal curve as the coating net charges alternated by the pH of BGE. The control of the EOF makes it possible to analyze different kinds of small molecules, peptides, and proteins successfully in the same capillary. Results showed that the stability and reproducibility for separations of fluoroquinolone standards were satisfactory for more than a hundred separations. A series of basic and acidic protein standards were separated with admirable efficiency and minimal adsorption using both polarities. The separation of tryptic BSA digest showed that the prepared capillary has immense potential in analyzing a single sample with both acidic and basic separations, which achieved the expectation in proteomics study by CE-MS.

DNAzyme-powered nucleic acid release from solid supports.

Here, we demonstrate use of a Mg2+-dependent, site-specific DNA enzyme (DNAzyme) to cleave oligos from polyacrylamide gel beads, which is suitable for use in drop-based assays. We show that cleavage efficiency is improved by use of a tandem-repeat cleavage site. We further demonstrate that DNAzyme-released oligos function as primers in reverse transcription of cell-released mRNA.

The interaction of poly(ethylenimine) with nucleic acids and its use in determination of nucleic acids based on light scattering.

For the first time, poly(ethylenimine) (PEI) was used to determine nucleic acids with a light scattering technique using a common spectrofluorometer. The interaction of PEI with DNA results in greatly enhanced intensity of light scattering at 300 nm, which is caused by the formation of the big particles between DNA and PEI. Based on this, a new quantitative method for nucleic acid determination in aqueous solutions has been developed. Under the optimum conditions, the enhanced intensity of light scattering is proportional to the concentration of nucleic acid in the range of 0.01-10.0 microg ml(-1) for herring sperm DNA (hsDNA), 0.02-10.0 microg ml(-1) for calf thymus DNA (ctDNA), 0.02-20.0 microg ml(-1) for yeast RNA (yRNA). The detection limits are 5.3, 9.9, and 13.7 ng ml(-1), respectively. Synthetic samples were determined satisfactorily. At the same time, the light scattering technique has been successfully used to obtain the information on the effects of pH and ionic strength on the formation and the stability of the DNA/PEI complex, which is important in some fields such as genetic engineering and gene transfer. Using ethidium bromide (EB) as a fluorescent probe, the binding of PEI with hsDNA was studied. Both the binding constant of EB with DNA and the number of binding sites per nucleotide decrease with increasing concentration of PEI, indicating noncompetitive inhibition of EB binding to DNA in the presence of PEI. And the association constant of PEI to DNA obtained is 1.2 x 10(5) M(-1). IR-spectra show that PEI interacts with DNA through both the phosphate groups and the bases of DNA and the formation of DNA/PEI complex may cause the change of the conformation of the DNA secondary structure, which is also proved by UV-spectra.

Amperometric hydrogen peroxide biosensor based on the immobilization of heme proteins on gold nanoparticles-bacteria cellulose nanofibers nanocomposite.

A novel matrix, gold nanoparticles-bacterial cellulose nanofibers (Au-BC) nanocomposite was developed for enzyme immobilization and biosensor fabrication due to its unique properties such as satisfying biocompatibility, good conductivity and extensive surface area, which were inherited from both gold nanoparticles (AuNPs) and bacterial cellulose nanofibers (BC). Heme proteins such as horseradish peroxidase (HRP), hemoglobin (Hb) and myoglobin (Mb) were successfully immobilized on the surface of Au-BC nanocomposite modified glassy carbon electrode (GCE). The immobilized heme proteins showed electrocatalytic activities to the reduction of H(2)O(2) in the presence of the mediator hydroquinone (HQ), which might be due to the fact that heme proteins retained the near-native secondary structures in the Au-BC nanocomposite which was proved by UV-vis and IR spectra. The response of the developed biosensor to H(2)O(2) was related to the amount of AuNPs in Au-BC nanocomposite, indicating that the AuNPs in BC network played an important role in the biosensor performance. Under the optimum conditions, the biosensor based on HRP exhibited a fast amperometric response (within 1s) to H(2)O(2), a good linear response over a wide range of concentration from 0.3 µM to 1.00 mM, and a low detection limit of 0.1 µM based on S/N=3. The high performance of the biosensor made Au-BC nanocomposite superior to other materials as immobilization matrix.

Biosensor arrays based on the degradation of thin polymer films interrogated by scanning photoinduced impedance microscopy.

Disposable sensors based on the degradation of thin films as a result of an enzymatic reaction have been developed into efficient enzyme detectors. Film degradation has traditionally been monitored using surface plasmon resonance (SPR), quartz crystal microbalance (QCM), or classical ac impedance measurements. The enzyme detection principle has now been integrated with an array technology derived from a recently developed impedance imaging technique, scanning photoinduced impedance microscopy (SPIM). SPIM is based on photocurrent measurements at field-effect structures. The material under investigation is commonly deposited onto a semiconductor-insulator substrate. In this work, field-effect capacitors were replaced by hydrogenated amorphous silicon (a-Si:H) n-i-p photodiode structures, which have recently been shown to be suitable for SPIM measurements with good lateral resolution. To demonstrate the feasibility of SPIM for the characterization of biosensor arrays, polymer dots of the inert polymer cellulose acetate and an alpha-chymotrypsin-sensitive poly(ester amide) were deposited onto a-Si:H n-i-p/SiO2 structures and their enzymatic degradation was monitored using a laser scanning setup.

Layer-by-layer self-assembly of multilayer films containing DNA and Eu3+: their characteristics and interactions with small molecules.

Thin films of alternating DNA and rare earth ion Eu3+ layers from dilute aqueous solutions were fabricated onto quartz substrates and silicon wafers through the layer-by-layer (LbL) self-assembly technique. UV-visible spectroscopy shows that a uniform layer of DNA can be fully adsorbed onto each alternate Eu3+ layer. Microscopic FTIR spectra show Eu3+ interacts with both the phosphate groups and nitrogenous bases of DNA, and the formation of [DNA/Eu]n films induces a change of the conformation of the DNA secondary structure to a certain extent. Various parameters affecting the DNA or Eu3+ loading into the composite film were investigated with emphasis on the effect of the pH and ionic strength of the DNA solution used for the film preparation. Atomic force microscopy was utilized to observe the morphologies of the DNA in the films obtained at two different pH values. Small molecules, such as alpha-tenoyltrifluoroacetone (TTA), Hoechst 33258 (Hoe), and ethidium bromide (EB), are all observed to interact with Eu3+ or DNA in the [DNA/Eu]n films. The [DNA/Eu]n films incorporated with these molecules show different fluorescent characteristics, and the fluorescence intensity of the films versus the bilayer number has a good linear relationship, confirming the potential for creating a different luminescence ability of the multilayer by controlling the number of DNA/Eu bilayers.