Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein-containing TaqMan probes.

The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland-Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways-both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.

Effect of Surface Adsorption on Temporal and Spatial Broadening in Micro Free Flow Electrophoresis.

Analyte adsorption onto surfaces presents a challenge for many separations, often becoming a significant source of peak broadening and asymmetry. We have shown that surface adsorption has no effect on peak position or spatial broadening in micro free flow electrophoresis (µFFE) separations. Surface adsorption does affect the time it takes an analyte to travel through the µFFE separation channel and therefore contributes to temporal broadening. These results were confirmed using µFFE separations of fluorescein, rhodamine 110, and rhodamine 123 in a low ionic strength buffer to promote surface adsorption. Peak widths and asymmetries were measured in both the temporal and spatial dimensions. Under these conditions rhodamine 123 exhibited significant interactions with the separation channel surface, causing increased peak broadening and asymmetry in the temporal dimension. Broadening or asymmetry in the spatial dimension was not significantly different than that of fluorescein, which did not interact with the capillary surface. The effect of strong surface interactions was assessed using µFFE separations of Chromeo P503 labeled myoglobin and cytochrome c. Myoglobin and cytochrome c were well resolved and gave rise to symmetrical peaks in the spatial dimension even under conditions where permanent adsorption onto the separation channel surface occurred.

Reversed-phase chromatography in extended-nano space for the separation of amino acids.

In this work we used reversed-phase chromatography in extended-nano channels to separate amino acids. A hydrophobic surface modification of extended-nano channels was established. A sample mixture of fluorescein and sulforhodamine B (0.5 and 0.05mM respectively) was used for the demonstration of a reversed-phase separation mode. A small amount of sample band (30fL) was injected into the separation channel, and two compounds were successfully separated. The maximum theoretical plate number of sulforhodamine B was 300,000plates/m. Two sets of 3 amino acids (3.75mM each) were separated using 0.01M citrate buffer (pH 5.5) with 0.01M sodium perchlorate and 12 and 25% of acetonitrile as a mobile phase. A successful separation (320,000plates/m with plate height of 3.2µm for serine) was accomplished.

Spectral study of fluorone dyes adsorption on chitosan-based polyelectrolyte complexes.

Polyelectrolyte complexes of the chitosan-chondroitin sulfate and chitosan-hyaluronate polycation-polyanion pairs were synthesized and characterized as potential dye adsorbents at different pH levels. Equilibrium isotherm analysis was applied to investigate the efficiency and the mechanism of the adsorption of fluorone dyes (fluorescein, eosin Y, erythrosin B) on the synthesized complexes. The inefficiency of the fluorescein adsorption was proved by two different quantitative spectroscopic methods. The adsorption isotherm for both eosin Y and erythrosin B was adequately described in terms of the Langmuir-Freundlich model. The observed room-temperature phosphorescence of the adsorbed erythrosin B was attributed to the surface inhomogeneity of the synthesized complexes. The revealed variation in the adsorption properties of fluorone dyes was related to the differences in their ionic forms as well as in their polarity and hydrophobicity.

[The synthetic growth medium Kings BS for detection of synthesis of fluorescein by bacteria Pseudomonas].

The synthetic growth medium KINGS BS was developed on the basis of substitution of proteose-peptone by L-glutamine or L-glutamine acid. The medium is used to detect the synthesis of fluorescein by bacteria Pseudomonas. The actual medium has equal diagnostic sensitivity and specificity as compared with medium King B. At the same time, the medium excels King B by content regularity and has no need of sterilization in vapor sterilizer and detects fluorescein in strains producing other pigments. The medium is suitable for standardization of technique of detection of synthesis of fluorescein by pseudomonads.

Milli-free flow electrophoresis: I. Fast prototyping of mFFE devices.

We coin a term of milli-free flow electrophoresis (mFFE) to describe mid-scale FFE with flow rates intermediate to macro-FFE and micro-FFE (µFFE). Introduced decades ago, mFFE did not find practical applications. We revive mFFE, as we view it as a viable purification complement to continuous synthesis in capillary reactors with product flow rates of ∼5 to 2000 µL/min, too small for macro-FFE but too large for µFFE. The development of the tandem of continuous synthesis/purification will require the production and evaluation of a large number of prototypes of mFFE devices. As the first step, we developed a fast (<24 h) and economical (∼$10) method for prototyping mFFE devices using a robotic milling machine. mFFE prototypes are constructed from two machined matching poly(methyl methacrylate) (PMMA) substrates, which are bonded in 10 min using dichloromethane to provide a strong and irreversible seal. Using the developed prototyping technology, we designed and evaluated 25 prototypes of mFFE devices. By optimizing the feed rates and rotational speeds of the drills, the depth of the electrode channels, the dimensions of the entrance and exit reservoirs, the sample flow rate, and the diameter and position of the sample input, we were able to achieve indefinitely long operation of the device with cycles of alternating 15-min electrophoresis and 0.5-min regeneration (bubble removal). The test analytes, rhodamine B and fluorescein, were baseline resolved by mFFE for flow rates ranging from 10 to 600 µL/min. These results prove that our prototyping approach is suitable for the challenging task of multi-parameter optimization of mFFE devices.

Femto liquid chromatography with attoliter sample separation in the extended nanospace channel.

A liquid chromatography system, comprising a separation column with a width and depth of a few hundred nanometers, was fabricated on a glass microchip (femto liquid chromatography, fLC). The size of this system was approximately 10(11) times smaller than that of a conventional LC system, the flow rate was subpicoliter/minute, and the injection volume was a few hundred attoliters. The fLC system did not require packing stationary phase and was capable of separating solutes with different molecular charges (fluorescein and sulforhodamine B) that could not be separated on a conventional LC column whose surface was covered with the same functional group as that of the column of the fLC system. The fLC system represented herein overcomes limitations of conventional chromatography separation, namely, heterogeneity of the stationary phases and eddy diffusion. Scale-down of the chromatography system brought advantages not only in reduction of sample volume but also in separation efficiency. The fLC system can analyze a very small amount of sample with high efficiency and will be useful in analyzing small samples, such as single cells and synaptic clefts. fLC greatly influences and benefits various fields such as life sciences, medicine, environmental science, and manufacturing by the improvement of separation technology.

Microsystem for field-amplified electrokinetic trapping preconcentration of DNA at poly(ethylene terephthalate) membranes.

In electrokinetic trapping (EKT), the electroosmotic velocity of a buffer solution in one area of a microfluidic device opposes the electrophoretic velocity of the analyte in a second area, resulting in transport of DNA to a location where the electrophoretic and electroosmotic velocities are equal and opposite and DNA concentrates at charged nanochannels. The method does not require an optical plug localization, a considerable advantage as compared to preconcentration techniques previously presented. In the work reported here, the trapping process is preceded by a field-amplification in the sample reservoir to reduce trapping time, as field-amplified EKT is shown to be an effective technique to preconcentrate samples from larger volumes. A theoretical model explaining the principle of field-amplified EKT considers different ionic strengths and cross-sectional areas in the microchip segments. The model is supported by experimental data using nucleic acids and fluorescein as sample analytes. An incorporated poly(ethylene terephthalate) (PET) membrane provides anion exclusion due to a negatively charged surface. A fluidic counter flow supports the trapping process in 100 nm pores due to anion exclusion. An analysis of Joule heating gives evidence that temperature gradient focusing effects are negligible and charge exclusion is responsible for trapping. The theoretical model developed and experimentally demonstrated can be exploited for the preconcentration of cell free fetal DNA circulating in maternal plasma and other rare nucleic acid species present in large sample volumes.

Characterization and performance of injection molded poly(methylmethacrylate) microchips for capillary electrophoresis.

Injection molded poly(methylmethacrylate) (IM-PMMA), chips were evaluated as potential candidates for capillary electrophoresis disposable chip applications. Mass production and usage of plastic microchips depends on chip-to-chip reproducibility and on analysis accuracy. Several important properties of IM-PMMA chips were considered: fabrication quality evaluated by environmental scanning electron microscope imaging, surface quality measurements, selected thermal/electrical properties as indicated by measurement of the current versus applied voltage (I-V) characteristic and the influence of channel surface treatments. Electroosmotic flow was also evaluated for untreated and O2 reactive ion etching (RIE) treated surface microchips. The performance characteristics of single lane plastic microchip capillary electrophoresis (MCE) separations were evaluated using a mixture of two dyes-fluorescein (FL) and fluorescein isothiocyanate (FITC). To overcome non-wettability of the native IM-PMMA surface, a modifier, polyethylene oxide was added to the buffer as a dynamic coating. Chip performance reproducibility was studied for chips with and without surface modification via the process of RIE with O2 and by varying the hole position for the reservoir in the cover plate or on the pattern side of the chip. Additionally, the importance of reconditioning steps to achieve optimal performance reproducibility was also examined. It was found that more reproducible quantitative results were obtained when normalized values of migration time, peak area and peak height of FL and FITC were used instead of actual measured parameters.

Parallel separation of multiple samples with negative pressure sample injection on a 3-D microfluidic array chip.

A simple and powerful microfluidic array chip-based electrophoresis system, which is composed of a 3-D microfluidic array chip, a microvacuum pump-based negative pressure sampling device, a high-voltage supply and an LIF detector, was developed. The 3-D microfluidic array chip was fabricated with three glass plates, in which a common sample waste bus (SW(bus)) was etched in the bottom layer plate to avoid intersecting with the separation channel array. The negative pressure sampling device consists of a microvacuum air pump, a buffer vessel, a 3-way electromagnet valve, and a vacuum gauge. In the sample loading step, all the six samples and buffer solutions were drawn from their reservoirs across the injection intersections through the SW(bus) toward the common sample waste reservoir (SW(T)) by negative pressure. Only 0.5 s was required to obtain six pinched sample plugs at the channel crossings. By switching the three-way electromagnetic valve to release the vacuum in the reservoir SW(T), six sample plugs were simultaneously injected into the separation channels by EOF and electrophoretic separation was activated. Parallel separations of different analytes are presented on the 3-D array chip by using the newly developed sampling device.

Field amplified sample stacking coupled with chip-based capillary electrophoresis using negative pressure sample injection technique.

A multi-T microchip for integrated field amplified sample stacking (FASS) with CE separation to increase the chip-based capillary electrophoresis (chip-based CE) sensitivity was developed. Volumetrically defined large sample plug was formed in one step within 5s by the negative pressure in headspace of the two sealed sample waste reservoirs produced using a syringe pump equipped with a 3-way valve. Stacking and separation can proceed only by switching the 3-way valve to release the vacuum in headspace of the two sample waste reservoirs. This approach considerably simplified the operations and the equipments for FASS in chip-based CE systems. Migration time precisions of 3.3% and 1.3% RSD for rhodamine123 (Rh123) and fluorescien sodium salt (Flu) in the separation of a mixture of Flu and Rh123 were obtained for nine consecutive determinations with peak height precisions of 4.8% and 3.4% RSD, respectively. Compared with the chip-based CE on the cross microchip, the sensitivity for analysis of FlTC, FITC-labeled valine (Val) and Alanine (Ala) increased 55-, 41- and 43-fold, respectively.

Affinity monoliths for ultrafast immunoextraction.

Affinity monoliths based on a copolymer of glycidyl methacrylate and ethylene dimethacrylate were developed for ultrafast immunoextractions. Rabbit immunoglobulin G (IgG) and anti-FITC antibodies were used as model ligands for this work. The antibody content of the monoliths was optimized by varying both the polymerization and immobilization conditions for preparing such supports. The temperature and porogen composition used during polymerization showed significant effects on monolith morphology and on the amount of antibodies that could be coupled to these materials. The effects of various immobilization procedures and coupling conditions were also evaluated, including the coupling temperature, pH, protein concentration, and use of high buffer concentrations. The maximum ligand density obtained for rabbit IgG was approximately 60 mg/g. When a 4.5 mm i.d. x 0.95 mm monolith disk containing anti-FITC antibodies was used, 95% extraction of fluorescein was achieved in 100 ms. These properties make such monoliths attractive for work in the rapid isolation of analytes from biological samples. Similar columns can be developed for other targets by varying the types of antibodies or binding agents placed within the monoliths.

Bias-free pneumatic sample injection in microchip electrophoresis.

We have developed a new microfluidic chip capable of accurate metering, pneumatic sample injection, and subsequent electrophoretic separation. The pneumatic injection scheme, enabling us to introduce a solution without sampling bias unlike electrokinetic injection, is based upon the hydrophobicity and wettability of channel surfaces. An accurately metered solution of 10 nL could be injected by pneumatic pressure into a hydrophilic separation channel through Y-shaped hydrophobic valves, which consist of polydimethylsiloxane (PDMS) and fluorocarbon (FC) film layers. We demonstrated the successful pneumatic injection of a red ink solution into the separation channel as a proof of the concept. A mixture of fluorescein and dichlorofluorescein (DCF) could be baseline-separated using a single power source in microchip electrophoresis.

Ultraviolet sealing and poly(dimethylacrylamide) modification for poly(dimethylsiloxane)/glass microchips.

Simple sealing methods for poly(dimethylsiloxane) (PDMS)/glass-based capillary electrophoresis (CE) microchips by UV irradiation are described. Further, we examined the possibility to modify the inner surface of separation channels, using polymethylacrylamide (PDMA) as a dynamic coating reagent. The surface properties of native PDMS, UV-irradiated PDMS, and PDMA-coated PDMS were systematically studied by atomic force microscopy (AFM), infrared absorption by attenuated total reflection infrared (ATR-IR) spectroscopy, and contact angle measurement. We found that PDMA forms a stable coating on PDMS and glass surfaces, eliminating the nonhomogeneous electroosmotic flow (EOF) in channels on PDMS/glass microchips, and improving the hydrophilicity of PDMS surfaces. Mixtures of flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and fluorescein were separated in 35 s using PDMA-coated PDMS/glass microchips. A high efficiency of theoretical plates with at least 1365 (105 000 N/m) and a good reproducibility with relative standard deviations (RSD) below 4% in five successive separations were achieved.

Integration of on-column immobilized enzyme reactor in microchip electrophoresis.

A simple method integrating an immobilized enzyme reactor into a microchip electrophoresis device was developed. The enzyme immobilization into a microchip was performed by spotting and drying a drop of dissolved nitrocellulose (NC) on a glass substrate, and adsorbing enzyme on the reconstituted NC membrane. This enzyme-immobilized glass plate was assembled with a polydimethylsiloxane substrate on which the separation channel was fabricated. The advantage of this method is the ability to easily change the position and size of the reactor within the microchip electrophoresis device. A beta-galactosidase reaction was demonstrated with fluorescein di-beta-D-galactopyranoside using this integrated on-column enzyme reactor. A successful electrophoretic separation of its hydrolysis products, i.e., fluorescein mono-beta-D-galactopyranoside (FMG) and fluorescein, was achieved. Enzyme kinetics and inhibition of the beta-galactosidase using FMG and 2-phenylethyl beta-D-thiogalactoside, respectively, were also studied with microchip electrophoresis.

A chip system for size separation of macromolecules and particles by hydrodynamic chromatography.

For the first time, a miniaturized hydrodynamic chromatography chip system has been developed and tested on separation of fluorescent nanospheres and macromolecules. The device can be applied to size characterization of synthetic polymers, biopolymers, and particles, as an attractive alternative to the classical separation methods such as size exclusion chromatography or field-flow fractionation. The main advantages are fast analysis, high separation efficiency, negligible solvent consumption, and easy temperature control. The prototype chip contains a rectangular flat separation channel with dimensions of 1 microm deep and 1000 microm wide, integrated with a 300-pL injector on a silicon substrate. The silicon microtechnology provides precisely defined geometry, high rigidity, and compatibility with organic solvents or high temperature. All flows are pressure driven, and a specific injection system is employed to avoid excessive sample loading times, demonstrating an alternative way of lab-on-a-chip design. Separations obtained in 3 min show the high performance of the device and are also the first demonstration of flat channel hydrodynamic chromatography in practice.

Lab-on-a-chip sample preparation using laminar fluid diffusion interfaces--computational fluid dynamics model results and fluidic verification experiments.

Microfluidic structures for the generation of laminar fluid diffusion interfaces (LFDIs) for sample preparation and analysis are discussed. Experimental data and the results of fluid modeling are shown. LFDIs are generated when two or more streams flow in parallel in a single microfluidic structure without any mixing of the fluids other than by diffusion of particles across the diffusion interface. It has been shown that such structures can be used for diffusion-based separation and detection applications. The method has been applied to DNA desalting, the extraction of small proteins from whole blood samples, and the detection of various constituents in whole blood, among other examples. In this paper the design and manufacture of self-contained microfluidic cartridges for the extraction of small molecules from a mixture of small and large molecules by diffusion is demonstrated. The cards are operated without any external instrumentation, and use hydrostatic pressure as the driving force. The performance of the cartridges is illustrated by separating fluorescein from a mixture of fluorescein and dextran of molecular weight 2 x 10(6). In a single pass, 98.6% of dextran was retained in the product whereas 43.1% of fluorescein was removed. The method is adjustable for different separation requirements, and computational fluid dynamics (CFD) models are shown that demonstrate the tuning of various microfluidic parameters to optimize separation performance. Other applications of LFDIs for establishment of stable concentration gradients, and the exposure of chemical constituents or biological particles to these concentration gradients are shown qualitatively. Microfluidic chips have been designed for high-throughput screening applications that enable the uniform and controlled exposure of cells to lysing agents, thus enabling the differentiation of cells by their sensitivity to specific agents in an on-chip cytometer coupled directly to the lysing structure.

An integrated fluorescence detection system in poly(dimethylsiloxane) for microfluidic applications.

This paper describes a prototype of an integrated fluorescence detection system that uses a microavalanche photodiode (microAPD) as the photodetector for microfluidic devices fabricated in poly(dimethylsiloxane) (PDMS). The prototype device consisted of a reusable detection system and a disposable microfluidic system that was fabricated using rapid prototyping. The first step of the procedure was the fabrication of microfluidic channels in PDMS and the encapsulation of a multimode optical fiber (100-microm core diameter) in the PDMS; the tip of the fiber was placed next to the side wall of one of the channels. The optical fiber was used to couple light into the microchannel for the excitation of fluorescent analytes. The photodetector, a prototype solid-state microAPD array, was embedded in a thick slab (1 cm) of PDMS. A thin (80 microm) colored polycarbonate filter was placed on the top of the embedded microAPD to absorb scattered excitation light before it reached the detector. The microAPD was placed below the microchannel and orthogonal to the axis of the optical fiber. The close proximity (approximately 200 microm) of the microAPD to the microchannel made it unnecessary to incorporate transfer optics; the pixel size of the microAPD (30 microm) matched the dimensions of the channels (50 microm). A blue light-emitting diode was used for fluorescence excitation. The microAPD was operated in Geiger mode to detect the fluorescence. The detection limit of the prototype (approximately 25 nM) was determined by finding the minimum detectable concentration of a solution of fluorescein. The device was used to detect the separation of a mixture of proteins and small molecules by capillary electrophoresis; the separation illustrated the suitability of this integrated fluorescence detection system for bioanalytical applications.