Biomedical subjects

Dehai Liang

Publications and source records attributed to Dehai Liang.

12 recordsLinked to original sources

PubMed2006

Designing polymer matrix for microchip-based double-stranded DNA capillary electrophoresis.

Polyacrylamide (PAM) was used as a model polymer to build up an empirical model that relates polymer molecular weight, polymer concentration and solution viscosity. The desired random copolymers of acrylamide (AM) and N,N-dimethylacrylamide (DMA) used as DNA separation media for different specifications were synthesized under the guidance of the empirical model. The separation performances of rationally designed copolymers were tested in a 1.2 cm long separation channel, simulating microchip-based capillary electrophoresis. pBR322/HaeIII digest was successfully separated with good separation resolution and fast speed. Validation of the sieving ability of our polymers was performed in the Agilent 2,100 Bioanalyzer. The results of the 10 bp (base pair) DNA ladder separation demonstrate the potential of our approach for the sieving matrix in microchip-based electrophoresis.

Acrylamides↗

PubMed2005

In vitro non-viral gene delivery with nanofibrous scaffolds.

Extracellular and intracellular barriers typically prevent non-viral gene vectors from having an effective transfection efficiency. Formulation of a gene delivery vehicle that can overcome the barriers is a key step for successful tissue regeneration. We have developed a novel core-shelled DNA nanoparticle by invoking solvent-induced condensation of plasmid DNA (beta-galactosidase or GFP) in a solvent mixture [94% N,N-dimethylformamide (DMF) + 6% 1x TE buffer] and subsequent encapsulation of the condensed DNA globule in a triblock copolymer, polylactide-poly(ethylene glycol)-polylactide (L8E78L8), in the same solvent environment. The polylactide shell protects the encapsulated DNA from degradation during electrospinning of a mixture of encapsulated DNA nanoparticles and biodegradable PLGA (a random copolymer of lactide and glycolide) to form a nanofibrous non-woven scaffold using the same solution mixture. The bioactive plasmid DNA can then be released in an intact form from the scaffold with a controlled release rate and transfect cells in vitro.

Animals↗

PubMed2005

Fractal behavior of functionalized fullerene aggregates. I. Aggregation of two-handed tetraaminofullerene with DNA.

In tris-buffered saline (TBS) with a trace of dimethylformamide (DMF), the homoaggregation process of a functionalized fullerene, the two-handed tetraaminofullerene (TH), and the heteroaggregation process (complex formation) of TH with DNA (pGL3-control plasmid) were studied dynamically by using a combination of static and dynamic laser light scattering measurements. Fractal behavior was investigated in the aggregation process of both TH homoaggregates and TH-DNA heteroaggregates. The stability of aggregates in solution depends on the molar concentration ratio R(M), defined as the molar ratio of moles of TH to moles of the DNA base pair. Higher R(M) values resulted in lower aggregate stability. The transition of the fractal dimension (Df) in TH homoaggregation by rapidly mixing 3.78 microM TH with an equal volume of the blank buffer was found to vary from a value of 1.46 to 2.02. Dynamic light scattering results revealed that, in the aggregation process, the change in the size distribution of aggregates with time could be related to a Df transition. In the Df transition region, the size distribution of homoaggregates displayed a drastic change from a single-mode distribution to a bimodal distribution, which clearly suggested a restructuring process with the formation of large aggregates. When the aggregation process finally reached equilibrium, Df = 2.02, the size of the homoaggregates had a single mode but a broad distribution. However, TH-DNA heteroaggregation showed a Df transition from 1.58 to 1.7, but over a shorter time range of less than 5 min. Then, the Df value fluctuated in the range of 1.7 and finally reached an equilibrium value of Df approximately 1.78, which was independent of molar concentration. There are two main action forces involved in the heteroaggregation process: van der Waals forces and attractive electrostatic forces, with the latter one being stronger and faster than that of the former. Therefore, a two-step action could occur in the heteroaggregation process. In the beginning of mixing, the attractive electrostatic forces dictated the aggregation process, and then van der Waals forces also got involved in the entire aggregation process. By using an initial concentration of 3.78 microM each and R(M) = 1, TH-DNA heteroaggregates showed more stable solution behavior than the homoaggregates. The lower Df value of the heteroaggregates could be related to a looser compact structure. Results from scanning electron microscopy (SEM) also disclosed the different textures between TH homoaggregates and TH-DNA heteroaggregates; the former had a more dense packing than the latter one.

DNA↗

PubMed2005

Quasi-interpenetrating network formed by polyacrylamide and poly(N,N-dimethylacrylamide) used in high-performance DNA sequencing analysis by capillary electrophoresis.

Quasi-interpenetrating network (IPN) formed by polyacrylamide and poly(N,N-dimethylacrylamide) was designed, synthesized, and tested as a high-performance DNA separation medium by capillary electrophoresis. The performance of quasi-IPN on DNA sequencing was determined by the acrylamide to dimethylacrylamide molar ratio, polyacrylamide molecular weight, and its size distribution. Under optimal operating conditions, quasi-IPN was able to achieve one-color DNA sequencing up to 1000 bases in 39 min, or 1200 bases in 60 min. Its performance was compared with some of the existing commercialized products, such as POP6 from Applied Biosystems and MegaBACE matrix from Amersham Biosciences. By using the ABI 310 Genetic Analyzer, even without optimized base-calling software, quasi-IPN yielded a read length of up to 700 bases of contiguous sequence (50-750 bases) in 35 min with 99.6% accuracy, or 750 bases of contiguous sequence (50-800 bases) in 37 min with 98.0% accuracy.

Acrylamides↗

PubMed2005

Fast separation of single-stranded oligonucleotides by capillary electrophoresis using OliGreen as fluorescence inducing agent.

The fast separation of oligonucleotide (oligos) sizing marker by CE using OliGreen and including effects due to the concentration of separation medium and urea denaturant is presented. OliGreen dye is found to be more sensitive than ethidium bromide (by a factor of about 6 based on S/N considerations) for the oligos' separations. Higher concentration of F127 in 1xTris-boricacid-EDTA (TBE) up to 30% w/v leads to better resolution of oligos separations. The addition of urea into the separation medium decreases the sensitivity. With an optimized running condition, the oligos sizing marker could be successfully separated with 1-base resolution within 1.3 min by using 30% w/v F127/1xTBE solution as the separation medium at an applied electric field of 800 V/cm in a 3 cm long capillary, the fastest capillary gel electrophoresis separation with high resolution reported to date for oligos in the similar size range.

Buffers↗

PubMed2004

Characterization of coliphage PR772 and evaluation of its use for virus filter performance testing.

Virus filtration is a key clearance unit operation in the manufacture of recombinant protein, monoclonal antibody, and plasma-derived biopharmaceuticals. Recently, a consensus has developed among filter manufacturers and end users about the desirability of a common nomenclature and a standardized test for classifying and identifying virus-retentive filters. The Parenteral Drug Association virus filter task force has chosen PR772 as the model bacteriophage to standardize nomenclature for large-pore-size virus-retentive filters (filters designed to retain viruses larger than 50 to 60 nm in size). Previously, the coliphage PR772 (Tectiviridae family) has been used in some filtration studies as a surrogate for mammalian viruses of around 50 to 60 nm. In this report, we describe specific properties of PR772 critical to the support of its use for the standardization of virus filters. The complete genomic sequence of virulent phage PR772 was determined. Its genome contains 14,946 bp with an overall G+C content of 48.3 mol%, and 32 open reading frames of at least 40 codons. Comparison of the PR772 nucleotide sequence with the genome of Tectiviridae family prototype phage PRD1 revealed 97.2% identity at the DNA level. By dynamic light-scattering analysis, its hydrodynamic diameter was measured as 82 +/- 6 nm, consistent with use in testing large-virus-retentive filters. Finally, dynamic light-scattering analysis of PR772 preparations purified on CsCl gradients showed that the phage preparations are largely monodispersed. In summary, PR772 appears to be an appropriate model bacteriophage for standardization of nomenclature for larger-pore-size virus-retentive filters.

Biotechnology↗

PubMed2003

Study of ethidium bromide effect on dsDNA separation by capillary zone electrophoresis and laser light scattering.

The effect of ethidium bromide (EB) on dsDNA separation was studied by using capillary zone electrophoresis (CZE) and laser light scattering (LLS). By increasing the concentration from 1 to 15 microg/mL, EB showed significant influence on the separation of pBR322 HaeIII in terms of resolution, speed, and signal-over-noise ratio. The physicochemical properties of DNA, such as mobility under electric field, charge-over-size ratio, and hydrodynamic radius, showed two declines, a sharp drop followed by a relaxed decrease, with increasing EB concentration. Such observations could be explained by using two modes of the dye binding process: intercalation and outside binding. The combination of results obtained from CZE and LLS also confirmed the existence of outside binding. Compared with intercalation, outside binding showed a weaker dye concentration dependence in neutralizing the negative charges on the DNA fragments. Static light scattering showed that and of DNA in 1xTris-borate-EDTA (TBE) were increased with increasing EB concentration. / of DNA with dye had a value larger than 1.5, suggesting a random coil conformation.

DNA↗

PubMed2002

Copolymer solutions as separation media for DNA capillary electrophoresis.

A review on copolymers used as DNA separation media in capillary electrophoresis is presented. Copolymers can combine the desirable properties of different monomers, yielding many attractive features, such as high sieving ability, low viscosity, self-assembly behavior and dynamic coating ability. Copolymers with different molecular architecture, including block copolymers, random copolymers, and graft copolymers, have been developed and tested as DNA separation media with unique and tailored properties that cannot be achieved easily by using only homopolymers.

DNA↗

PubMed2002

Separation of double-stranded DNA fragments by capillary electrophoresis using polyvinylpyrrolidone and poly(N,N-dimethylacrylamide) transient interpenetrating network.

A noncross-linked interpenetrating polymer network (IPN), consisting of poly(N,N-dimethylacrylamide) (PDMA) and polyvinylpyrrolidone (PVP, weight-average molecular weight M(w) = 1 x 10(6) g/mol) was synthesized by polymerizing N,N-dimethylacrylamide (DMA) monomers directly in PVP buffer solution and tested as a separation medium for double-stranded (ds)DNA analysis without further purification. Due to the incompatibility of PVP and PDMA, a simple solution mixture could incur a microphase separation and showed poor performance on dsDNA separation. However, a dramatic improvement was achieved by the formation of an IPN. We attributed the high sieving ability of IPN as due to an increase in the number of entanglements by the more extended polymer chains. Apparent viscosity studies showed that the IPN had a much higher viscosity than the simple mixture containing the same amount of PDMA and PVP. In 1 x Tris-borate-EDTA (TBE) buffer, the concentration ratio of PDMA and PVP had a great effect on the DNA separation. At optimal conditions, the 22 fragments in pBR322/HaeIII DNA were successfully separated within 15 min, with a resolution of better than 1.0 for 123/124 bp.

Acrylamides↗

PubMed2002

Concentration gradient used in double-stranded DNA separation by capillary electrophoresis.

Effects of concentration gradient on double-stranded DNA (dsDNA) separation by capillary electrophoresis are presented. By using a concentration gradient in the range between 0.8% and 3.2% for poly(N,N-dimethylacrylamide) (PDMA), the presence of a mesh-size gradient in the capillary could enhance the separation of larger size DNA fragments, better than that based on a single uniform concentration over the same capillary length. A decrease in the column length could make the gradient effect more obvious. An optimal capillary length could be achieved by using a judicious combination of the concentration gradient and the concentration range, yielding a maximum resolution for the system. The standard deviation of the migration time measured for each DNA fragment was less than 5% in ten continuous runs, suggesting that the gradient formed inside the column was quite stable.

Acrylamides↗

PubMed2002

Coupling of optical characterization with particle and network synthesis for biomedical applications.

Polymeric microspheres containing a magnetic core have been used in cancer therapy for biophysical targeting of antitumor agents and in magnetic resonance imaging as contrasting agents. For the Human Genome Project, deoxyribose nucleic acid (DNA) capillary electrophoresis has become the most widely used analytical technique where a key component is the design of an effective separation medium. The synthesis and optical characterization of polymeric coated superparamagnetic nanoparticles and of (self-assembled) polymer networks by means of a range of physical techniques, including laser light scattering and laser-induced fluorescence detection, are presented. (1) Polymeric microspheres with a superparamagnetic core. A water-in-oil microemulsion approach has been used successfully to synthesize the superparamagnetic core and the polymeric microsphere in one continuous step. The synthesis permits us to control the magnetic nanoparticle size and the thickness of the hydrogel, ranging from 80 to 320 nm. Magnetite concentration in the microspheres, calculated by vibrating-sample magnetometry, was found to be up to 3.3 wt %. The internal structure of the microspheres, as observed by atomic force microscopy, confirmed a core-shell model. (2) Development of new separation media for DNA capillary electrophoresis. Block copolymers in selective solvents can self-assemble to form supramolecular structures in solution. The nanostructures can be characterized in the dilute concentration regime by means of laser light scattering. At semidilute concentrations, the mesh size, the supramolecular structure, and the surface morphology can be investigated by means of small angle x-ray scattering and atomic force microscopy. The structural knowledge and the information on chain dynamics can then be correlated with electrophoresis using laser-induced fluorescence detection to provide a deeper understanding for the development of new separation media.

Biophysical Phenomena↗

PubMedDate not supplied

Nanostructures of complexes formed by calf thymus DNA interacting with cationic surfactants.

Synchrotron small-angle X-ray scattering was used to study the nanostructures of the complexes formed by calf thymus DNA interacting with cationic lipids (or surfactants) of didodecyldimethylammonium bromide (DDAB), cetyltrimethylammonium bromide (CTAB), and their mixture with a zwitterionic lipid of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (PHGPC). The effects of lipid/DNA ratios, DNA chain flexibility, lipid topology, and neutral lipid mixing on the nanostructures of DNA-lipid complexes were investigated. The complexes between double-stranded DNA (dsDNA) and double-tailed DDAB formed a bilayered lamellar structure, whereas the complexes between dsDNA and single-tailed CTAB preferred a structure of 2D hexagonal close packing of cylinders. With single stranded DNA (ssDNA) interacting with CTAB, the complexes showed a Pm3n cubic structure due to the different chain flexibility between dsDNA and ssDNA. The lipid molecules bound by rigid dsDNA like to form cylindrical micelles, whereas lipids bound to flexible ssDNA could form spherical or short cylindrical micelles. The addition of the neutral single-chained PHGPC lipids to the CTAB lipids could induce a structural transition of dsDNA-lipid complexes from a 2D hexagonal to a multi-bilayered lamellar structure. The parallel DNA strands were intercalated in the water layers of lamellar stacks of the mixed lipid bilayers. The DNA-DNA spacing depended on the ratios of charged lipid to neutral lipid, and charged lipid to DNA, respectively.

Animals↗