Integrated isotachophoretic preconcentration with zone electrophoresis separation on a quartz microchip for UV detection of flavonoids

A quartz microchip integrated isotachophoretic (ITP) preconcentration with zone electrophoresis (ZE) separation was fabricated using a novel multi-point pressure method featured in normal temperature and lower pressure during bonding process. ITP followed by subsequential ZE of two flavonoids, quercetin and isorhamnetin on the microchip was performed consecutively on the homemade microfluidic workstation with UV detection, resulting in a decreased detectable concentration of 32-fold, compared to the ZE mode only, and their detection limits decreased down to 0.2 microg/mL and 1.2 microg/mL, respectively.     

Solid supports for micro analytical systems

The development of micro analytical systems requires that fluids are able to interact with the surface of the microfluidic chip in order to perform analysis such as chromatography, solid phase extraction, and enzymatic digestion. These types of analyses are more efficient if there are solid supports within the microfluidic channels. In addition, solid supports within microfluidic chips are useful in producing devices with multiple functionalities. In recent years there have been many approaches introduced for incorporating solid supports within chips. This review will explore several state of the art methods and applications of introducing solid supports into chips. These include packing chips with beads, incorporating membranes into chips, creating supports using microfabrication, and fabricating gels and polymer monoliths within microfluidic channels.     

Velocity-difference induced focusing of xanthine and purine metabolites by capillary electrophoresis using a dynamic pH junction

Summary Velocity-difference induced focusing (V-DIF) of analytes by a dynamic pH junction represents a simple yet effective on-line preconcentration method to improve concentration sensitivity in capillary electrophoresis (CE). Differences in buffer type, pH and conductivity between sample and background electrolyte (BGE) segments of the capillary are properties used to optimize purine focusing within a multi-section electrolyte system. This method permits the injection of large volumes of sample (up to 450 nL or about 18% of capillary length), resulting in over a 50-fold improvement in sensitivity with baseline resolution. The limit of detection (S/N=3) for xanthine is determined to less than 4.0×10⁻⁸ M under optimum conditions when using UV detection. Analysis of micromolar amounts of xanthine in pooled urine is also demonstrated without sample pretreatment. A dual mechanism involving dynamic pH and isotachophoretic modes is proposed to enhance analyte focusing performance when employing buffer pH junctions based on different types of electrolyte co-ions.     

Fabrication of silver-coated silica microspheres through mussel-inspired surface functionalization

This paper reports a droplet-based microfluidic device composed of patterned co-planar electrodes in an all-in-a-single-plate arrangement and coated with dielectric layers for electrowetting-on-dielectric (EWOD) actuation of discrete droplets. The co-planar arrangement is preferred over conventional two-plate electrowetting devices because it provides simpler manufacturing process, reduced viscous drag, and easier liquid-handling procedures. These advantages lead to more versatile and efficient microfluidic devices capable of generating higher droplet speed and can incorporate various other droplet manipulation functions into the system for biological, sensing, and other microfluidic applications. We have designed, fabricated, and tested the devices using an insulating layer with materials having relatively high dielectric constant (SiO(2)) and compared the results with polymer coatings (Cytop) with low dielectric constant. Results show that the device with high dielectric layer generates more reproducible droplet transfer over a longer distance with a 25% reduction in the actuation voltage with respect to the polymer coatings, leading to more energy efficient microfluidic applications. We can generate droplet speeds as high as 26 cm/s using materials with high dielectric constant such as SiO(2).     

A split microchannel design and analytical model to compensate for electroosmotic instabilities in micro-separations

Organic polymers offer many advantages as materials for the construction of microfluidic devices but suffer frequently from the limitation that the electrodynamic flow they support can exhibit considerable instability. This article describes a split-channel microfluidic device that can be used to compensate for changes in electroosmotic flow. The design of the separation system divides an analyte plug after injection between two separation channels of differing length. The two channels are later recombined for single point detection, eliminating the need for a scanning optical detection system. The utility of this simple design lies in the fact that the migration time of any analyte can be referenced to its twin in the parallel separation channel. This eliminates the need for a separate electroosmotic marker and allows mobilities measured in multiple devices to be compared quantitatively. Using a model adopted from the literature, the data from the split channel system can be used to precisely account for the drift that characterizes electrophoretic separations made in a polymer chip. The relative standard deviations of the analyte mobilities measured for replicate runs on multiple devices were reduced from values as high as 20% to ca. 1% RSD. This internal standardization procedure also appears to address other sources of drift in the electroosmotic flow (EOF) supported by the polymer microchannel, eliminating the need for careful monitoring of either the temperature or reservoir pH between separation runs.     

Sacrificial layer microfluidic device fabrication methods

Over the past 15 years, research in the field of microfluidics has experienced rapid growth due to significant potential advantages such as low cost, short analysis times, and elimination of sources of contamination. Although etched and thermally bonded glass substrates have seen widespread use and offer solid performance, device fabrication still remains cumbersome. Recent advances in sacrificial layer microfabrication methods for microfluidics have overcome many disadvantages of conventional fabrication approaches. Phase-changing sacrificial layers have been implemented in making inexpensive and high-performance polymer microchips for electrophoretic analysis, protein focusing, and sample preconcentration. In addition, novel channel fabrication methods based on standard thin-film processes, which are readily integratable with microfabrication techniques used for electrical components, are being applied increasingly for the creation of microfluidic devices. These new sacrificial layer fabrication approaches will be instrumental in making low-cost and high-quality polymer microchips, and in interfacing electrical and fluidic systems on glass or semiconductor substrates.     

Preconcentration and separation of double-stranded DNA fragments by electrophoresis in plastic microfluidic devices

We have evaluated double-stranded DNA separations in microfluidic devices which were designed to couple a sample preconcentration step based on isotachophoresis (ITP) with a zone electrophoretic (ZE) separation step as a method to increase the concentration limit of detection in microfluidic devices. Developed at ACLARA BioSciences, these LabCard trade mark devices are plastic 32 channel chips, designed with a long sample injection channel segment to increase the sample loading. These chips were designed to allow stacking of the sample into a narrow band using discontinuous ITP buffers, and subsequent separation in the ZE mode in sieving polymer solutions. Compared to chip ZE, the sensitivity was increased by 40-fold and we showed baseline resolution of all fragments in the PhiX174/HaeIII DNA digest. The total analysis time was 3 min/sample, or less than 100 min per LabCard device. The resolution for multiplexed PCR samples was the same as obtained in chip ZE. The limit of detection was 9 fg/microL of DNA in 0.1xpolymerase chain reaction (PCR) buffers using confocal fluorescence detection following 488 nm laser excitation with thiazole orange as the fluorescent intercalating dye.     

Photochromic soft materials: flavylium compounds incorporated into pluronic F-127 hydrogel matrixes

The performance of flavylium-based photochromic systems is increased by their incorporation into Pluronic F-127 matrixes, which switch from polymeric solutions to micelles to gels with changes in temperature depending on copolymer concentration. Two flavylium compounds, 7,4'-dihydroxyflavylium and 7-(N,N-diethylamino)-4-hydroxyflavylium, both exhibiting a small thermal cis-trans isomerization barrier in water were investigated. In the first system the flavylium in the gel photoswitches from the colorless trans-chalcone (Ct) species to the yellow flavylium cation (AH+) with quantum yield Phi=0.04 (25 degrees C) at pH 2.2 or to the orange quinoidal base (A) with quantum yield Phi=0.015 (25 degrees C) at pH 5.2. The photoproducts revert back to their initial form by a thermal process characterized by first-order kinetics; the rate constants exhibit a bell shape variation with pH, with a maximum at pH 4.3 (lifetime 4.2 min). The second system, 7-(N,N-diethylamino)-4-hydroxyflavylium, does not exhibit photochemistry in water but, when incorporated into the Pluronic F-127 gel, switches from yellow to red with a quantum yield of Phi=0.01 at pH 4.9. The respective thermal back reaction takes place with a lifetime of 66.7 min1. The flavylium network of chemical reactions is a good sensor for the detection of not only the critical micelle temperature but also the gelation temperature of Pluronic and like solutions and, in some instances, the exposure to UV and visible radiation.     

Sample preconcentration from dilute solutions on micro/nanofluidic platforms: A review

Biochemical detection plays a critical role in many analytical fields. For example, blood samples include many proteins with relevance to disease diagnosis and therapeutic monitoring. Foods and beverages contain a large number of chemicals and compounds which must be quantified and characterized to ensure their compliance with safety standards. Detecting trace amounts of contaminants in ambient air or water samples is essential in monitoring the environment and protecting human health. Therefore, effective techniques for performing the rapid and reliable detection of targeted analytes are required. Compared to conventional macroscale devices, microfluidic systems have many advantages, including a greater sensitivity, a faster response time, a reduced sample and reagent consumption, and a greater portability. Accordingly, many microfluidic systems for sample detection have been proposed in recent years. The performance of such devices relies on the target analyte being present in a sufficient concentration to enable its detection. In many biomedical, food testing and environmental applications, the detection limit was restricted. Thus, the sample must first be concentrated before the detection process is carried out. Accordingly, this review provides a comprehensive review of recent advances for sample preconcentration with emphasis on utilizing ion concentration polarization (ICP) effects in micro/nanofluidics platforms. We start with a brief introduction regarding the importance of preconcentration using micro/nanofluidics platforms, followed by in‐depth discussions of the ICP effects for the preconcentration and applications to biomedical analysis, food testing and environmental monitoring. Finally, the article concludes with a brief perspective on the future development of the field.     

Harnessing Joule heating in microfluidic thermal gel electrophoresis to create reversible barriers for cell enrichment

Gel electrophoresis is a ubiquitous bioanalytical technique used to characterize the components of cell lysates. However, analyses of bulk lysates sacrifice detection sensitivity because intracellular biomolecules become diluted, and the liberation of proteases and nucleases can degrade target analytes. This report describes a method to enrich cells directly within a microfluidic gel as a first step toward online measurement of trace intracellular biomolecules with minimal dilution and degradation. Thermal gels were employed as the gel matrix because they can be reversibly converted between liquid and solid phases as a function of temperature. Rather than fabricate costly heating elements into devices to control temperature—and thus the phase of the gel—Joule heating was used instead. Adjoining regions of liquid-phase and solid-phase gel were formed within microfluidic channels by selectively inducing localized Joule heat. Cells migrated through the liquid gel but could not enter the solid gel—accumulating at the liquid–solid gel boundary—whereas small molecule contaminants passed through to waste. Barriers were then liquified on-demand by removing Joule heat to collect the purified, non-lysed cells for downstream analyses. Using voltage-controlled Joule heating to regulate the phase of thermal gels is an innovative approach to facilitate in-gel cell enrichment in low-cost microfluidic devices.     

On-line preconcentration of organic anions in capillary electrophoresis by solid-phase extraction using latex-coated monolithic stationary phases

Quaternary ammonium functionalised polymeric latex particles were coated onto the wall of a fused-silica capillary or onto a methacrylate monolithic bed synthesised inside the capillary in order to create ion-exchange stationary phases of varying ion-exchange capacity. These capillaries were coupled in-line to a separation capillary and used for the solid-phase extraction (SPE), preconcentration and subsequent separation of organic anions by capillary electrophoresis. A transient isotachophoretic gradient was used for the elution of bound analytes from the SPE phase using two modes of separation. The first comprised a low capacity SPE column combined with a fluoride/octanesulfonate discontinuous electrolyte system in which peak compression occurred at the isotachophoretic gradient front. The compressed anions were separated electrophoretically after elution from the SPE preconcentration phase and resolution was achieved by altering the pH of the electrolyte in which the separation was performed. In the second approach, a latex-coated monolithic SPE preconcentration stationary phase was used in combination with a fluoride/perchlorate electrolyte system, which allowed capillary electrochromatographic separation to occur behind the isotachophoretic gradient front. This method permitted the removal of weakly bound anions from the SPE phase, thereby establishing the possibility of sample clean-up. The effect of the nature of the strong electrolyte forming the isotachophoretic gradient on the separation and also on the preconcentration step was investigated. Capillary electrochromatography of inorganic and organic species performed on the latex-coated monolithic methacrylate column highlighted the presence of mixed-mode interactions resulting from the incomplete coverage of latex particles onto the monolithic surface. Analyte preconcentration prior to separation resulted in compression of the analyte zone by a factor of 300. Improvement in the limit of detection of up to 10400 times could be achieved when performing the preconcentration step and the presented methods had limits of detection (S/N=3) ranging between 1.5 and 12 nM for the organic anions studied.     

A Selective Solid-Phase Extraction and Preconcentration Method with Using Molecularly Imprinted Polymer for Piroxicam in Pharmaceutical Sample

Abstract

A powerful sample cleanup procedure using molecularly imprinted polymer as a solid phase extraction material for the preconcentration of piroxicam in pharmaceutical samples has been developed. The optimized conditions for the preparation of molecularly imprinted polymer as a selective sorbent for the preconcentration of this drug were also studied. It is very important to note that a preconcentration factor of 1000 and retention capacity of 30.0 mg/g can be achieved. Recoveries up to 95% were accomplished for the analyte of interest from a pharmaceutical piroxicam capsule sample. All of these experiments were also performed with nonimprinted polymer.     

Measuring the transverse concentration gradient between adjacent laminar flows in a microfluidic device by a laser-based refractive index gradient detector

A detection scheme that probes the refractive index gradient (RIG) between adjacent laminar flows in microfluidic devices has been developed and evaluated. The behavior of low Reynolds number flows has been well documented and shows that molecular transport (mixing) between adjacent laminar flows occurs by molecular diffusion between the flow boundaries. A diode laser has been used to probe the transverse concentration gradient at a selected position along a microchannel. The concentration gradient is affected by the transverse diffusion from a flow with analyte into a flow initially without analyte. To optimize sensitivity, the RIG is probed at a position in which molecular diffusion across the boundary of the two flows has been minimal, i.e. just after the flow initially without analyte merges with the flow initially containing the analyte at a given concentration. The RIG formed causes the laser beam, impinging orthogonal to the RIG through the microchannel, to be deflected. The angle of deflection is then monitored on a position sensitive detector (PSD). Currently, this detection scheme is demonstrated to provide quantitative detection of sucrose, as a test analyte, with a concentration limit of detection (LOD) of 96 ppm (w/v) or 280 muM, corresponding to 1.3x10(-5) DeltaRI units using 3sigma baseline noise. A dynamic range of 96 ppm to 50% sucrose is obtained. This detection method provides universal detection selectivity for microfluidic analysis systems that are becoming increasingly useful in monitoring chemical systems, particularly for the polymer, pharmaceutical and life sciences fields. For a larger molecular weight analyte with a smaller diffusion coefficient, lower concentration and RI LODs were achieved since detection sensitivity is a function of analyte diffusion. For example, for the polymer poly (ethylene glycol) with a molar mass of 11 840 g mol(-1), the LOD was experimentally determined to be 56 ppm (4.7 muM), equivalent to a RI LOD of 4.5x10(-6) DeltaRI (3sigma). The detection limit for proteins was also found to be favorable. For example, with the current configuration, ribonuclease A (RNAse) had a LOD of 46 ppm (3.4 muM), and bovine serum albumin (BSA) had a LOD of 54 ppm (780 nM).     

Fluorescence of commercial Pluronic F127 samples: Temperature-dependent micellization

We present a novel approach of using the butylated hydroxytoluene (BHT) antioxidant found in commercial Pluronic F127 samples as a marker of polymer aggregation. The BHT marker was compared to the pyrene dye and static light scattering methods as a way to measure the critical micelle concentration (CMC) and critical micelle temperature (CMT). The n→π(?) transitions of BHT are sensitive to the microenvironment as demonstrated by plotting the fractional intensities of its excitation (≈280nm) and emission (≈325nm) peaks. BHT is more sensitive to changes in temperature than concentration. The partition coefficient increases ≈40-fold for pyrene compared to ≈2-fold for BHT when the temperature is increased from 25 to 37°C. CMT values determined using the BHT fluorescence decrease with increasing F127 concentration. Our results show that BHT can be used as a reliable marker of changes in the microenvironment of Pluronic F127.     

On-chip sample pretreatment using a porous polymer monolithic column for solid-phase microextraction and chemiluminescence determination of catechins in green tea

A porous polymer monolithic column for solid-phase microextraction and chemiluminescence detection was integrated into a simple microfluidic chip for the extraction and determination of catechins in green tea. The porous polymer was prepared by poly(glycidyl methacrylate-co-ethylene dimethacrylate) and modified with ethylenediamine. Catechins can be concentrated in the porous polymer monolithic column and react with potassium permanganate to give chemiluminescence. The microfluidic chip is reusable with high sensitivity and very low reagent consumption. The on-line preconcentration and detection can be realized without an elution step. The enrichment factor was calculated to be about 20 for catechins. The relative chemiluminescence intensity increased linearly with concentration of catechin from 5.0 × 10(-9) to 1.0 × 10(-6) M and the limit of detection was 1.0 × 10(-9) M. The proposed method was applied to determine catechin in green tea. The recoveries are from 90% to 110% which benefits the actual application for green tea samples.     

Electrokinetic trapping and concentration enrichment of DNA in a microfluidic channel

We report a simple and efficient method for enriching the concentration of charged analytes within microfluidic channels. The method relies on exerting spatial control over the electrokinetic velocity of an analyte. Specifically, the electroosmotic (eo) velocity of the buffer solution in one region of the microfluidic system opposes the electrophoretic (ep) velocity of the analyte in the other region. This results in ep transport of DNA to the location where the ep and eo velocities are equal and opposite. Accumulation of the analyte occurs at this location. This enrichment method is conceptually distinct from field-amplification stacking, isotachophoresis, micelle sweeping, size exclusion, and other methods that have been previously reported. The method requires no complex microfabricated structures, no special manipulation of the solvent, and the enriched analyte remains in solution rather than being captured on a solid support. A concentration enrichment factor of 800 can be achieved for 20mer DNA in a fluidic channel having dimensions of 100 mum x 25 mum x 5 mm. The time required to achieve this level of enrichment is 300 s, and the enriched zone has a minimum width of 100 mum.     

Electrokinetic concentration enrichment within a microfluidic device using a hydrogel microplug

A simple and efficient approach for concentration of charged molecules in microfluidic devices is described. The functional component of the system is a hydrogel microplug photopolymerized within the main channel of a microfluidic device. When an appropriately biased voltage is applied across the hydrogel, charged analyte molecules move from the source well toward the hydrogel. Transport of the analyte through the hydrogel is slow compared to its velocity in the microfluidic channel, however, and therefore it concentrates at the hydrogel/solution interface. For an uncharged hydrogel, a bias of 100 V leads to a approximately 500-fold enrichment of the DNA concentration within 150 s, while the same conditions result in an enrichment of only 50-fold for fluorescein. Somewhat lower enrichment factors are observed when a negatively charged hydrogel is used. A qualitative model is proposed to account for the observed behavior.     

Electrochemical control of solid phase micro-extraction: conducting polymer coated film material applicable for preconcentration/analysis of neutral species

Exploitation of the physical, chemical and electrically conductive properties of poly(3-dodecylthiophene) (P3DDT) for the preconcentration and release in solid phase microextraction (SPME) of organometallic arsenobetaine (AsB) from aqueous media was investigated. Hydrophobic interactions between this neutral arsenic species and an undoped polythiophene (no applied potential) with n-substituted alkyl groups (n=12) in the three position were used for the diffusion-controlled preconcentration. After absorption into the polymer matrix, the chemical properties of this conductive polymer were changed by applying an external potential. This potential provides a sufficient driving force for desorption of the analyte from the extraction phase into an aqueous solution for subsequent analysis. The applied positive potential oxidizes the polymer to its charged hydrophilic state, which releases the neutral analyte. The concentration and speciation of the analyte from the sample matrix was analyzed by HPLC coupled to an ICP-MS. The diffusion-controlled uptake was fast (equilibrium attained within minutes) and did not require pretreatment of the analyte. The electrochemically-controlled release of the analyte is also very rapid (within minutes). This conducting polymer film system, therefore, can offer analytical applications for the convenient preconcentration and subsequent analysis of neutral environmental species.     

Antitumor effect of pluronic F-127 gel containing mitomycin C on sarcoma-180 ascites tumor in mice.

Pluronic F-127 (PLF-127) gels were evaluated as a sustained-release vehicle for intraperitoneal administration of mitomycin C (MMC) in order to enhance the therapeutic effects of MMC against a Sarcoma-180 ascites tumor in mice. Tumor cell injections were made on day 0 and injections of MMC in 25% (w/w) PLF-127 on day 1, both intraperitoneally. A prolongation of the life span of tumor-bearing mice following injection of therapeutic PLF-127 was noted, and PLF-127 containing MMC was therapeutically more active than free drug. The high chemotherapeutic efficiency of MMC in PLF-127 was striking at high doses, which would be toxic in the case of the drug alone. PLF-127 gels exhibit reverse thermal behavior and are fluid at refrigerator temperature, but are soft gels at body temperature. The in vitro release experiments indicated that Pluronic gel might serve as a rate-controlling barrier and be useful as a vehicle for sustained-release preparations of MMC to be administered intraperitoneally. These results suggest that sustained-release occurs in the peritoneum and that effective drug concentrations can be maintained by the preparation.     

Temperature gradient focusing of bio-analyte in a microfluidic channel dealing with non-Newtonian electrolyte considering temperature-dependent zeta potential

Temperature gradient focusing (TGF) relies on establishing a precise balance between the electrophoretic motility of a target analyte and the advective flow of the background electrolyte (BGE) to locally concentrate the analyte in a microfluidic configuration. This paper presents a finite-element-based numerical analysis where the coupled electric field and the transport equations are solved to describe the effects of the shear-dependent apparent viscosity of a non-Newtonian BGE on the localized concentration buildup of a charged bio-sample inside a microchannel by TGF via Joule heating. Effects of the temperature-dependent nature of the wall zeta potential and the flow behavior index (n) of BGE on the flow, thermal, and species concentration profiles inside the microchannel have been investigated. Study using a fluorescein-Na analyte sample shows that the maximum normalized analyte concentration (C_max/C₀) reduces as the zeta potential increases linearly with temperature. The maximum concentration enhancement is achieved when the BGE displays the Newtonian rheology. For example, C_max/C₀ increases 134- to 280-fold when n is increased from 0.8 to 1 (pseudoplastic regime) and again reduces to 190-fold when n increases further from 1 to 1.2 (dilatant regime).