Capillary gel affinity electrophoresis of DNA fragments.

The incorporation of an affinity ligand within a polyacrylamide gel provides a general means of manipulating the selectivity of capillary gel electrophoresis separations. As an example of this approach, high resolution of DNA restriction fragments by capillary gel affinity electrophoresis has been achieved by adding a soluble intercalating agent, ethidium bromide, to the gel-buffer system. A migration model has been developed that can be used for selectivity optimization. Various parameters, such as ligand concentration and applied electric field, have been examined in terms of their influence on retention and selectivity of different-size DNA molecules. From this study, high-resolution separations have been developed with efficiencies as high as 10(7) theoretical plates per meter.     

Optimization of ammonia absorption refrigeration process: effects of seasonal ambient temperature fluctuations

A flow-sheet simulation program has been developed to optimize the absorption refrigeration process for ammonia. This program takes economic considerations into account in addition to the thermodynamic parameters and also allows for seasonal fluctuations in ambient temperature. The program is faster to run than the currently available non-linear programs and can be used for the preliminary design of ammonia absorption refrigeration plants.     

Anisotropic elasticity of magnetically ordered agarose gel

The physical properties of agarose gel prepared under strong magnetic fields were investigated. The storage modulus was measured by the reflection method with an ultrasonic pulse. The measurement results of the gel''s elasticity indicate that agarose gel has anisotropic properties. The elasticity and its anisotropy depend on the concentration of the gel and the magnetic field to which it is exposed. The experimental results indicate that the anisotropic network structure of the gel is induced by the exposure to the magnetic field during gelation. The gelation mechanism under a magnetic field is discussed.     

Anisotropic elasticity of magnetically ordered agarose gel

Abstract

The physical properties of agarose gel prepared under strong magnetic fields were investigated. The storage modulus was measured by the reflection method with an ultrasonic pulse. The measurement results of the gel's elasticity indicate that agarose gel has anisotropic properties. The elasticity and its anisotropy depend on the concentration of the gel and the magnetic field to which it is exposed. The experimental results indicate that the anisotropic network structure of the gel is induced by the exposure to the magnetic field during gelation. The gelation mechanism under a magnetic field is discussed.     

Combination of fiber-guided pulsed erbium and holmium laser radiation for tissue ablation under water

Because of the high absorption of near-infrared laser radiation in biological tissue, erbium lasers and holmium lasers emitting at 3 and 2 μm, respectively, have been proven to have optimal qualities for cutting or welding and coagulating tissue. To combine the advantages of both wavelengths, we realized a multiwavelength laser system by simultaneously guiding erbium and holmium laser radiation by means of a single zirconium fluoride (ZrF(4)) fiber. Laser-induced channel formation in water and poly(acrylamide) gel was investigated by the use of a time-resolved flash-photography setup, while pressure transients were recorded simultaneously with a needle hydrophone. The shapes and depths of vapor channels produced in water and in a submerged gel after single erbium and after combination erbium-holmium radiation delivered by means of a 400-μm ZrF(4) fiber were measured. Transmission measurements were performed to determine the amount of pulse energy available for tissue ablation. The effects of laser wavelength and the delay time between pulses of different wavelengths on the photomechanical and photothermal responses of meniscal tissue were evaluated in vitro by the use of histology. It was observed that the use of a short (200-μs, 100-mJ) holmium laser pulse as a prepulse to generate a vapor bubble through which the ablating erbium laser pulse can be transmitted (delay time, 100 μs) increases the cutting depth in meniscus from 450 to 1120 μm as compared with the depth following a single erbium pulse. The results indicate that a combination of erbium and holmium laser radiation precisely and efficiently cuts tissue under water with 20-50-μm collateral tissue damage.     

All-optical switching in plant blue light photoreceptor phototropin

We theoretically analyze all-optical switching in the recently characterized LOV2 domain from Avena sativa (oat) phot1 phototropin, a blue-light plant photoreceptor, based on nonlinear intensity-induced excited-state absorption. The transmission of a cw probe laser beam at 660 nm corresponding to the peak absorption of the first excited L-state, through the LOV2 sample, is switched by a pulsed pump laser beam at 442 nm that corresponds to the maximum initial D state absorption. The switching characteristics have been analyzed using the rate equation approach, considering all the three intermediate states and transitions in the LOV2 photocycle. It is shown that for a given pump pulse intensity, there is an optimum pump pulsewidth for which the switching contrast is maximum. It is shown that the probe laser beam can be completely switched off (100% modulation) by the pump laser beam at 50 kW/cm² for a concentration of 1 mM with sample thickness of 5.5 mm. The switching characteristics are sensitive to various parameters such as concentration, rate constant of L-state, peak pump intensity and pump pulse width. At typical values, the switch-off and switch-on time is 1.6 and 22.3 mus, respectively. The switching characteristics have also been used to design all-optical not and the universal nor and nand logic gates     

Group velocity control of a light pulse using giant nonlinearities

In this paper, we propose and demonstrate a scheme to enhance nonlinearities of a probe pulse at both cross-phase modulation (XPM) and self-phase modulation (SPM) in a four-level system. Based on standing wave grating generated by counter-propagating resonant signal fields and an additional off-resonant coupling field, a giant nonlinear refractive index of the resonant probe field is obtained with absorption suppressed. Group velocity of the probe pulse can be controlled by both XPM and SPM nonlinearities.     

Concentration of DNA in a flowing stream for high-sensitivity capillary electrophoresis

A novel sample pretreatment device is described, and its application to the concentration and purification of crude DNA samples in a flowing stream for subsequent capillary electrophoresis is demonstrated. The device consists of two gap junctions, each covered with a conductive membrane material and built upon a flow channel made of PEEK tubing. Upon the application of an electric field between the junctions, the negatively charged DNA fragments can resist the hydrodynamic flow stream and are trapped between the junctions. DNA fragments dissolved in microliter volumes are captured in a nanoliter-sized band by simply pushing the sample solution through the device. Depending on their electrophoretic mobility, other interfering materials in a crude sample can be removed from the trapped DNA fragments by washing. The selective permeability of the membrane to small ions allows efficient desalting. The concentrated and purified DNA fragments are released by simply turning off or reversing the electric field. Recovery is up to 95%. Performance of the device was evaluated using crude products of fluorescent dye-primer cycle-sequencing reactions. Compared to these crude reaction products, samples purified in the capture device and subsequently collected showed dramatically enhanced signal and resolution when run on a conventional capillary-electrophoresis instrument. Furthermore, the device could be connected in-line to a capillary system for direct injection. The device has great potential for enabling lab-on-a-chip systems to be used with real-world samples.     

Development of rocket electrophoresis technique as an analytical tool in preformulation study of tetanus vaccine formulation

Rocket Electrophoresis (RE) technique relies on the difference in charges of the antigen and antibodies at the selected pH. The present study involves optimization of RE run conditions for Tetanus Toxoid (TT). Agarose gel (1% w/v, 20 ml, pH 8.6), anti-TT IgG - 1 IU/ml, temperature 4-8°C and run duration of 18 h was found to be optimum. Height of the rocket-shaped precipitate was proportional to TT concentration. The RE method was found to be linear in the concentration range of 2.5 to 30 Lf/mL. The method was validated and found to be accurate, precise, and reproducible when analyzed statistically using student's t-test. RE was used as an analytical method for analyzing TT content in plain and marketed formulations as well as for the preformulation study of vaccine formulation where formulation additives were tested for compatibility with TT. The optimized RE method has several advantages: it uses safe materials, is inexpensive, and easy to perform. RE results are less prone to operator's bias as compared to flocculation test and can be documented by taking photographs and scanned by densitometer; RE can be easily standardized for the required antigen concentration by changing antitoxin concentration. It can be used as a very effective tool for qualitative and quantitative analysis and in preformulation studies of antigens.     

Efficient generation of ultra broadband supercontinuum in a mid-infrared field

We theoretically investigate the generation of ultra broadband supercontinuum from helium atoms exposed to a linearly polarized mid-infrared field. By adopting a UV trigger pulse to the mid-infrared field, the continuous harmonic yields are significantly enhanced by 3.5 orders, and a supercontinuum with the width of 230?eV is observed. The spectrum can support a sub-20 as pulse, which is below one atomic unit of time (24 as). The short quantum path is selected by adjusting the time delay between the UV pulse and the mid-infrared pulse, then broadband single 70 as pulses with tunable central wavelengths are obtained, which can be extended to the ‘water window’ region (284–543?eV).     

Stimuli-responsive releasing of gold nanoparticles and liposomes from aptamer-functionalized hydrogels

Controlled release of therapeutic agents is important for improving drug efficacy and reducing toxicity. Recently, hydrogels have been used for controlled release applications. While the majority of the previous work focused on releasing the cargo in response to physical stimuli such as temperature, light, electric field, and pH, we aim to trigger cargo release in the presence of small metabolites. In our system a DNA aptamer that can bind to adenosine, AMP, and ATP was used as a linker to attach either DNA-functionalized gold nanoparticles or liposomes to DNA-functionalized hydrogels. In the presence of the metabolite, both the nanoparticle and liposome cargos were released. The effect of salt, temperature, target concentration, and drying has been systematically studied. Interestingly, we found that the gel can be completely dried while retaining the DNA linkages and adenosine induced release was still achieved after rehydration. Our work demonstrates that aptamers can be used to control the release of drugs and other materials attached to hydrogels.     

A reversible gel for chiral separations

The use of a guanosine gel as a chiral selector in capillary electrophoresis is introduced. Guanosine gels are reversible organized media that are formed through the self-association of guanosine compounds. Their degree of organization and their physicochemical properties can be modulated through variations in guanosine monomer concentration, pH, temperature, and cation content. Baseline resolution of the d and l enantiomers of propranolol was achieved using a reversible biogel formed by 5'-guanosine monophosphate as the run buffer in capillary electrophoresis. Conditions were optimized to provide enantiomeric resolution of 2.1-2.3 in less than 5 min. The reversibility of the gel network offers potential advantages for chiral separations, including the possibility of using thermal or chemical dissociation of the gel network to remove the nucleoside monomers from the separated enantiomers, thereby eliminating the chiral selector as a source of physical contamination of the enantiomerically pure products and spectral background in UV absorbance detection.     

Quantitation of transgenic Bt event-176 maize using double quantitative competitive polymerase chain reaction and capillary gel electrophoresis laser-induced fluorescence

In this work, a new procedure useful to quantitatively analyze genetically modified organisms (GMOs) in foods is described and applied to analyze transgenic Bt Event-176 maize. The method developed consists of coamplifications of specific DNA maize sequences with internal standards using quantitative competitive PCR (QC-PCR). The QC-PCR products are quantitatively analyzed using a capillary gel electrophoresis (CGE) with laser-induced fluorescence detection (LIF) method developed at our laboratory that utilizes a physically adsorbed coating. The CGE-LIF procedure allows the use of internal standards differing by only 10 bp from the original target fragments, to our knowledge, the smallest size difference that can be found in the bibliography for QC-PCR of GMOs. A spectrofluorometric procedure using ROX reference dye is proposed to solve calibration problems of input DNA concentration. It is demonstrated that the use of ROX drastically enhances the accuracy of the quantitative analysis by QC-PCR. Reproducibility of analysis times and corrected peak areas (measured as target/competitor PCR products ratio) for the CGE-LIF separations are determined to be better than 0.91 and 1.93% (RSD, n = 15) respectively, for three different days. It is shown that CGE-LIF provides better resolution and a signal/noise ratio improvement of approximately 700-fold compared to slab gel electrophoresis. The good possibilities in terms of quantitative analysis of GMOs provided by this new method are confirmed by determining the Bt Event-176 maize content in certified reference maize powder and food samples of known composition. This procedure opens the possibility for accurate quantitation of multiple GMOs in a single run.     

Femtosecond single-shot correlation system: a time-domain approach

We introduce, analyze, and experimentally demonstrate what to the best of our knowledge is a new pulse correlation technique that is capable of real-time conversion of a femtosecond pulse sequence into its spatial image. Our technique uses a grating at the entrance of the system, thus introducing a transverse time delay (TTD) into the transform-limited reference pulse. The shaped signal pulses and the TTD reference pulse are mixed in a nonlinear optical crystal (LiB(3)O(5)), thus producing a second-harmonic field that carries the spatial image of the temporal shaped signal pulse. We show that the time scaling of the system is set by the magnification of the anamorphic imaging system as well as by the grating frequency and that the time window of the system is set by the size of the grating aperture. Our experimental results show a time window of ~20 ps. We also show that the chirp information of the shaped pulse can be recovered by measurement of the spectrum of the resulting second-harmonic field.     

Development of Rocket Electrophoresis Technique as an Analytical Tool in Preformulation Study of Tetanus Vaccine Formulation

ABSTRACT

Rocket Electrophoresis (RE) technique relies on the difference in charges of the antigen and antibodies at the selected pH. The present study involves optimization of RE run conditions for Tetanus Toxoid (TT). Agarose gel (1% w/v, 20 ml, pH 8.6), anti-TT IgG – 1 IU/ml, temperature 4–8°C and run duration of 18 h was found to be optimum. Height of the rocket-shaped precipitate was proportional to TT concentration. The RE method was found to be linear in the concentration range of 2.5 to 30 Lf/mL. The method was validated and found to be accurate, precise, and reproducible when analyzed statistically using student's t-test. RE was used as an analytical method for analyzing TT content in plain and marketed formulations as well as for the preformulation study of vaccine formulation where formulation additives were tested for compatibility with TT. The optimized RE method has several advantages: it uses safe materials, is inexpensive, and easy to perform. RE results are less prone to operator's bias as compared to flocculation test and can be documented by taking photographs and scanned by densitometer; RE can be easily standardized for the required antigen concentration by changing antitoxin concentration. It can be used as a very effective tool for qualitative and quantitative analysis and in preformulation studies of antigens.     

Quantum-interference and the concentration of Er3+ ion effect on left-handedness with zero absorption and large negative refractive index in Er3+:YAG crystal

The electromagnetically induced left-handedness with zero absorption and large negative refractive index was investigated in a solid Er³⁺:YAG crystal with a four-level system proposed for an atomic medium. It was found that the frequency region with simultaneous negative permittivity and negative permeability, the zero absorption intervals, and the maximum values of the negative refractive index can be adjusted by changing the signal field, the coherent field, as well as the concentration of Er³⁺ ion in crystal. It is shown that wider zero absorption intervals with a higher index of refraction can be easily obtained when the signal field is only off resonance. The slab fabricated by the left-handed solid medium Er³⁺:YAG crystal with zero absorption may be a practical candidate for designing perfect lenses.     

Fluorescence lifetime for on-the-fly multiplex detection of DNA restriction fragments in capillary electrophoresis

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.     

Highly sensitive pyrosequencing system with polymer-supported enzymes for high-throughput DNA analysis

A highly sensitive massively parallel pyrosequencing system employing a gel matrix to immobilize enzymes at high density in microreaction chambers is demonstrated. Reducing the size of microreaction chambers in a DNA analyzer is important to achieve a high throughput utilizing a commercially available detection device or camera. A high-performance system can be attained by detecting signals from one reaction chamber with one photopixel of around several micrometers by utilizing a 1:1 image magnification. However, the use of small beads immobilizing DNA has a disadvantage in detecting luminescence because only small amounts of DNA can be immobilized on the bead surfaces for sequencing. As luminescence intensity could be enhanced by increasing the luciferase density in the chambers, we overcame this difficulty by using a gel matrix to immobilize luciferase at a high concentration in the microreaction chambers. Luminescence 1 order of magnitude higher could be observed with the new method compared to the conventional method. Consequently, the chamber size and bead size immobilizing DNA could be reduced to as small as 6.5 and 4 μm, respectively. This can be successfully applied to achieving small, inexpensive, pyrosequencing systems with high throughput.     

DNA-functionalized monolithic hydrogels and gold nanoparticles for colorimetric DNA detection

Highly sensitive and selective DNA detection plays a central role in many fields of research, and various assay platforms have been developed. Compared to homogeneous DNA detection, surface-immobilized probes allow washing steps and signal amplification to give higher sensitivity. Previously research was focused on developing glass or gold-based surfaces for DNA immobilization; we herein report hydrogel-immobilized DNA. Specifically, acrydite-modified DNA was covalently functionalized to the polyacrylamide hydrogel during gel formation. There are several advantages of these DNA-functionalized monolithic hydrogels. First, they can be easily handled in a way similar to that in homogeneous assays. Second, they have a low optical background where, in combination with DNA-functionalized gold nanoparticles, even ～0.1 nM target DNA can be visually detected. By using the attached gold nanoparticles to catalyze the reduction of Ag+, as low as 1 pM target DNA can be detected. The gels can be regenerated by a simple thermal treatment, and the regenerated gels perform similarly to freshly prepared ones. The amount of gold nanoparticles adsorbed through DNA hybridization decreases with increasing gel percentage. Other parameters including the DNA concentration, DNA sequence, ionic strength of the solution, and temperature have also been systematically characterized in this study.     

Large-Area Arrays of Polymer-Tethered Gold Nanorods with Controllable Orientation and Their Application in Nano-Floating-Gate Memory Devices

In this work, it is reported that large-area (centimeter-scale) arrays of non-close-packed polystyrene-tethered gold nanorod (AuNR@PS) can be prepared through a liquid–liquid interfacial assembly method. Most importantly, the orientation of AuNRs in the arrays can be controlled by changing the intensity and direction of electric field applied in the solvent annealing process. The interparticle distance of AuNR can be tuned by varying the length of polymer ligands. Moreover, the AuNR@PS with short PS ligand are favorited to form orientated arrays with the assistance of electric field, while long PS ligands make the orientation of AuNRs difficult. The orientated AuNR@PS arrays are employed as the nano-floating gate of field-effect transistor memory device. Tunable charge trapping and retention characteristics in the device can be realized by electrical pulse with visible light illumination. The memory device with orientated AuNR@PS array required less illumination time (1 s) at the same onset voltage in programming operation, compared to the control device with disordered AuNR@PS array (illumination time: 3 s). Moreover, the orientated AuNR@PS array-based memory device can maintain the stored data for more than 9000 s, and exhibits stable endurance characteristic without significant degradation in 50 programming/reading/erasing/reading cycles.