Temperature Gradient Focusing Research Articles

Development of aptamer‐based affinity assays using temperature gradient focusing: Minimization of the limit of detection

A method is described for an aptamer-based affinity assay using a combination of two nonconventional techniques, temperature gradient focusing (TGF) and field-amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Human immunodeficiency virus reverse transcriptase (HIVRT) is used as the protein target for the assay. The TGF and FACSI-TGF assays are compared to similar results obtained with conventional CE. A range of starting aptamer concentrations are used to determine the optimal LOD for human immunodeficiency virus reverse transcriptase (HIVRT) using each approach. The results indicate that the LODs for HIVRT obtained with TGF and FACSI-TGF are comparable to or even lower than the LODs obtained with conventional CE in spite of the inferior detector used for the TGF and FACSI-TGF assays (arc lamp and low-cost CCD for TGF versus LIF with PMT for CE). It is hypothesized that this is due to the greater reproducibility of the TGF and FACSI-TGF techniques since they do not employ a defined sample injection. The lowest LOD achieved with the new aptamer assay approach is more than an order of magnitude lower than that reported for a similar CE-based aptamer assay for the same target.

Development of a temperature gradient focusing method for in situ extraterrestrial biomarker analysis

Scanning temperature gradient focusing (TGF) is a recently described technique for the simultaneous concentration and separation of charged analytes. It allows for high analyte peak capacities and low LODs in microcolumn electrophoretic separations. In this paper, we present the application of scanning TGF for chiral separations of amino acids. Using a mixture of seven carboxyfluorescein succinimidyl ester-labeled amino acids (including five chiral amino acids) which constitute the Mars7 standard, we show that scanning TGF is a very simple and efficient method for chiral separations. The modulation of TGF separation parameters (temperature window, pressure scan rate, temperature range, and chiral selector concentration) allows optimization of peak efficiencies and analyte resolutions. The use of hydroxypropyl-beta-CD at low concentration (1-5 mmol/L) as a chiral selector, with an appropriate pressure scan rate ( -0.25 Pa/s) and with a low temperature range (3-25 degrees C over 1 cm) provided high resolution between enantiomers (Rs >1.5 for each pair of enantiomers) using a short, 4 cm long capillary. With these new results, the scanning TGF method appears to be a viable method for in situ trace biomarker analysis for future missions to Mars or other solar system bodies.

Numerical modeling of Joule heating‐induced temperature gradient focusing in microfluidic channels

Temperature gradient focusing (TGF) is a recently developed technique for spatially focusing and separating ionic analytes in microchannels. The temperature gradient required for TGF can be generated either by an imposed temperature gradient or by Joule heating resulting from an applied electric field that also drives the flow. In this study, a comprehensive numerical model describing the Joule heating induced temperature development and TGF is developed. The model consists of a set of governing equations including the Poisson-Boltzmann equation, the Laplace equation, the Navier-Stokes equations, the energy equations and the mass transport equation. As the thermophysical and electrical properties including the liquid dielectric constant, viscosity, and electric conductivity are temperature-dependent, these governing equations are coupled, and therefore the coupled governing equations are solved numerically by using a CFD-based numerical method. The numerical simulations agree well with the experimental results, suggesting the valid mathematical model presented in this study.

Finite sample effect in temperature gradient focusing

Temperature gradient focusing (TGF) is a new and promising equilibrium gradient focusing method which can provide high concentration factors for improved detection limits in combination with high-resolution separation. In this technique, temperature-dependent buffer chemistry is employed to generate a gradient in the analyte electrophoretic velocity. By the application of a convective counter-flow, a zero-velocity point is created within a microchannel, at which location the ionic analytes accumulate or focus. In general, the analyte concentration is small when compared with buffer ion concentrations, such that the focusing mechanism works in the ideal, linearized regime. However, this presumption may at times be violated due to significant sample concentration growth or the use of a low-concentration buffer. Under these situations the sample concentration becomes non-negligible and can induce strong nonlinear interactions with buffer ions, which eventually lead to peak shifting and distortion, and the loss of detectability and resolution. In this work we combine theory, simulation, and experimental data to present a detailed study on nonlinear sample-buffer interactions in TGF. One of the key results is the derivation of a generalized Kohlrausch regulating function (KRF) that is valid for systems in which the electrophoretic mobilities are not constant but vary spatially. This generalized KRF greatly facilitates analysis, allowing reduction of the problem to a single equation describing sample concentration evolution, and is applicable to other problems with heterogeneous electrophoretic mobilities. Using this sample evolution equation we have derived an understanding of the nonlinear peak deformation phenomenon observed experimentally in TGF. We have used numerical simulations to validate our theory and to quantitatively predict TGF. Our simulation results demonstrate excellent agreement with experimental data, and also indicate that the proper inclusion of Taylor dispersion is important for the accurate modeling of TGF. This work is an important first step towards the understanding and prediction of the more complex, nonlinear, and multi-species interactions which often occur in on-chip electrophoretic assays such as TGF.

Ballistic dispersion in temperature gradient focusing

Molecular dispersion is caused by both molecular diffusion and non-uniform bulk fluid motion. While the Taylor–Aris dispersion regime is the most familiar regime in microfluidic systems, an oft-overlooked regime is that of purely kinematic (or ballistic) dispersion. In most microfluidic systems, this dispersion regime is transient and quickly gives way to Taylor–Aris dispersion. In electrophoretic focusing methods such as temperature gradient focusing (TGF), however, the characteristic time scales for dispersion are fixed, and focused peaks may never reach the Taylor limit. In this situation, generalized Taylor dispersion analysis is not applicable. A heuristic model is developed here which accounts for both molecular diffusion and advective dispersion across all dispersion regimes, from pure diffusion to Taylor dispersion to pure advection. This model is compared to results from TGF experiments and accurately captures both the initial decrease and subsequent increase in peak widths as electric field strength increases. The results of this combined analytical and experimental study provide a useful tool for estimation of dispersion and optimization of TGF systems.

Temperature gradient focusing in a PDMS/glass hybrid microfluidic chip

This paper reports the application of temperature gradient focusing (TGF) in a PDMS/glass hybrid microfluidic chip. With TGF, by the combination of a temperature gradient along a microchannel, an applied electric field, and a buffer with a temperature-dependent ionic strength, analytes are focused by balancing their electrophoretic velocities against the bulk velocity of the buffer containing the analytes. In this work, Oregon Green 488 carboxylic acid was concentrated approximately 30 times as high as the initial concentration in 45 s at moderate electric strength of 70 V/cm and a temperature gradient of 55 degrees C across the PDMS/glass hybrid microfluidic chip with a 1 cm long capillary.

Counterflow Rejection of Adsorbing Proteins for Characterization of Biomolecular Interactions by Temperature Gradient Focusing

A new technique is described for the analysis of small molecules in samples containing serum proteins and for the measurement of the binding of small molecules to serum proteins. The new technique is based on temperature gradient focusing (TGF) and takes advantage of the counterflow used with TGF to exclude serum proteins from the analysis channel while small molecules are focused for detection. The technique is demonstrated for the measurement of the binding constant between a small molecule and serum albumin using both a direct measurement of the free fraction of the small molecule as well as using a competitive binding assay.

Temperature Gradient Focusing with Field-Amplified Continuous Sample Injection for Dual-Stage Analyte Enrichment and Separation

We describe the serial combination of temperature gradient focusing (TGF) and field-amplified continuous sample injection (FACSI) for improved analyte enrichment and electrophoretic separation. TGF is a counterflow equilibrium gradient method for the simultaneous concentration and separation of analytes. When TGF is implemented with a low conductivity sample buffer and a (relatively) high conductivity separation buffer, a form of sample enrichment similar to field-amplified sample stacking (FASS) or field-amplified sample injection (FASI) is achieved in addition to the normal TGF sample enrichment. FACSI-TGF differs from FASI in two important respects: continuous sample injection, versus a discrete injection, is utilized; because of the counterflow employed for TGF, the stacking interface exists in a pseudo-stationary region outside of the separation column. Notably, analyte concentration enrichment factors greater than the ratio of separation and sample conductivities (gamma) were achieved in this method. For gamma=6.1, the concentration factor for one model analyte (Oregon Green 488) was found to be 36-fold higher with FACSI-TGF as compared to TGF without FACSI. A separation of five fluorescently labeled amino acids is also demonstrated with the technique, yielding an average enrichment of greater than 1000-fold.

Taylor–Aris dispersion in temperature gradient focusing

Microfluidic temperature gradient focusing (TGF) uses an axial temperature gradient to induce a gradient in electrophoretic flux within a microchannel. When balanced with an opposing fluid flow, charged analytes simultaneously focus and separate according to their electrophoretic mobilities. We present a theoretical and experimental study of dispersion in TGF. We model the system using generalized dispersion analysis that yields a 1-D convection-diffusion equation that contains dispersion terms particular to TGF. We consider analytical solutions for the model under uniform temperature gradient conditions. Using a custom TGF experimental setup, we compare focusing measurements with the theoretical predictions. We find that the theory well represents the focusing behavior for flows within the Taylor-Aris dispersion regime.

Simultaneous Concentration and Separation of Coumarins Using a Molecular Micelle in Micellar Affinity Gradient Focusing

We report the use of a molecular micelle for the simultaneous separation and concentration of neutral and hydrophobic analytes using micellar affinity gradient focusing (MAGF). The technique, MAGF, combines the favorable features of micellar electrokinetic chromatography and temperature gradient focusing. The focusing of neutral coumarin analytes was accomplished by the use the molecular micelle, poly(sodium undecenyl sulfate) (poly-SUS). Concentration enhancements of 10-25-fold/min were achieved for focusing of the coumarin dyes. The effect of varying the temperature gradient on the resolution of two of the coumarin dyes was also investigated, demonstrating that improved resolution could be achieved by reducing the steepness of the temperature gradient. In addition, with scanning-mode MAGF (in which the peaks are sequentially scanned past a fixed detection point by varying the buffer counterflow velocity), the use of poly-SUS was shown to produce repeatable and quantitative analyte peaks, making quantitative separations possible with the MAGF technique. Finally, it was shown that peak areas could be increased in scanning MAGF by reducing the scan rate so that the sensitivity of the method can be adjusted as needed.

Theoretical and numerical analysis of temperature gradient focusing via Joule heating

We present a detailed theoretical and numerical analysis of temperature gradient focusing (TGF) via Joule heating-an analytical species concentration and separation technique relying upon the dependence of an analyte's velocity on temperature due to the temperature dependence of a buffer's ionic strength and viscosity. The governing transport equations are presented, analyzed, and implemented into a quasi-1D numerical model to predict the resulting temperature, velocity, and concentration profiles along a microchannel of varying width under an applied electric field. Numerical results show good agreement with experimental trials presented in previous work. The model is used to analyze the effects of varying certain geometrical and experimental parameters on the focusing performance of the device. Simulations also help depict the separation capability of the device, as well as the effectiveness of different buffer systems used in the technique. The analysis provides rule-of-thumb methodology for implementation of TGF into analytical systems, as well as a fundamental model applicable to any lab-on-a-chip system in which Joule heating and temperature-dependent electrokinetic transport are to be analyzed.

Temperature gradient focusing for microchannel separations

Microfluidic devices are currently evolving beyond a concept technology into a mature field, with examples already evident in commercial applications. Microchips employing free solution electrophoresis have benefited from over 20 years of development in capillary electrophoresis (CE) aimed at enhancing selectivity and sensitivity. Like conventional CE, these devices suffer from relatively poor detection limits because of the extremely small quantities of analyte that are injected onto the separation column and the very short (10–100 μm) optical detection path lengths. These issues become exacerbated in microfluidics, which can have integrated injection schemes allowing for even smaller injection plugs than traditionally afforded, as well as exceedingly small cross-sectional channel areas (for a review, see [1]). Additionally, many of the microchip substrate materials, whether glass-based or polymeric, can have high backgrounds for fluorescence or other optical detection methods and so degrade detection limits relative to fused silica employed in conventional CE. Sensitivity enhancements have been realized through instrumental detection improvements, novel detection geometries, and detection methods other than fluorescence or absorbance. Several improvements in sample preconcentration prior to the separation step have also evolved to improve detection limits, including sample stacking methods, sweeping methods, and isotachophoresis, as well as chromatographic methods. Another group of analyte concentration methods have been termed equilibrium gradient focusing methods, with the most prevalent example in microchannels being isoelectric focusing, where the separation arises from variations in analyte isoelectric points along a pH gradient. Gradient methods combine concentration and separation steps by forcing analytes to a unique equilibrium point along the separation axis; analytes focus at their unique point of null velocity, with the separation based upon analytes having differing equilibrium points. Due to the nature of the focusing, peaks become both narrower and more concentrated throughout the separation, allowing for high resolution and sensitivity. A novel new equilibrium method was introduced for proteins by Koegler and Ivory using a variable electric field [2, 3]. Electric field gradient focusing (EFGF) has been further developed in recent years by the groups of Lee and Woolley [4, 5], as well as by Myers and Bartle [6]; at present the method has been successfully integrated into microfluidic devices [7–9]. As an alternative to pH or electric field gradients, a temperature gradient can be employed, as has been shown with temperature gradient focusing (TGF), which is based upon balancing analytes’ electrophoretic mobilities against a bulk flow containing both hydrodynamic and electroosmotic flow components (Fig. 1). TGF has been applied to a wide variety of analytes, including proteins, DNA, small dye molecules, and amino acids, in both capillary and microfluidic formats (Fig. 2; [10–16]; Kamande MW, Ross D, Locascio LE, Warner IM, 2006, submitted to Anal Chem; Huber DE, Santiago JG, 2006, submitted to Electrophoresis). Anal Bioanal Chem (2007) 387:155–158 DOI 10.1007/s00216-006-0913-4

Low-Power Concentration and Separation Using Temperature Gradient Focusing via Joule Heating

We present an experimental study of temperature gradient focusing (TGF) exploiting an inherent Joule heating phenomenon. A simple variable-width PDMS device delivers rapid and repeatable focusing of model analytes using significantly lower power than conventional TGF techniques. High electric potential applied to the device induces a temperature gradient within the microchannel due to the channel's variable width, and the temperature-dependent mobility of the analytes causes focusing at a specific location. The PDMS device also shows simultaneous separation and concentration capability of a mixture of two sample analytes in less than 10 min. An experiment combining Joule heating with external heating/cooling further supports the hypothesis that temperature is indeed the dominant factor in achieving focusing with this technique.

Scanning Temperature Gradient Focusing

Temperature gradient focusing (TGF) is a recently developed technique for the simultaneous concentration and electrophoretic separation of ionic analytes in microfluidic channels. One drawback to TGF as it has previously been described is the limited peak capacity; only a small number of analyte peaks (approximately 2-3) can be simultaneously focused and separated. In this paper, we report on a variation of the TGF method whereby the bulk flow rate is varied over time so that a large number of analytes can be sequentially focused, moved past a fixed detection point, and flushed to waste. In addition to improved peak capacity, the detection limits of the scanning TGF method can be adjusted on-the-fly, as needed for different samples. Finally, scanning TGF provides a technique by which high-resolution, high-peak-capacity electrophoretic separations can be performed in simple, straight, and short microfluidic channels.

Quantitative temperature gradient focusing performed using background electrolytes at various pH values

Temperature gradient focusing (TGF) has previously been shown to be a practical technique for simultaneous concentration and separation of a variety of samples. In this paper, we demonstrate that TGF can be conducted at a wide range of pH values. Techniques for first-order prediction of the suitability of a given BGE for focusing are discussed. Buffer suitability for TGF is assessed experimentally by simultaneously concentrating and separating a pair of fluorescent analytes. One analyte is held at constant concentration for use as an internal standard while the concentration of the other dye is varied. Peak area is shown to vary linearly with the input dye concentration. A high degree of resolution (R(s) >3) of fluorescein and carboxyfluorescein, as well as for two LysoSensor-based dyes, is also observed. Foucusing and separation by TGF was successfully conducted quantitatively in BGE solutions of pH from 3.0 to 10.5.

Simultaneous Concentration and Separation of Enantiomers with Chiral Temperature Gradient Focusing

A new technique is demonstrated for the simultaneous concentration and high-resolution separation of chiral compounds. With temperature gradient focusing, a combination of a temperature gradient, an applied electric field, and a buffer with a temperature-dependent ionic strength is used to cause analytes to move to equilibrium, zero-velocity points along a microchannel or capillary. Different analytes are thus separated spatially and concentrated in a manner that resembles isoelectric focusing but that is applicable to a greater variety of analytes including small chiral drug molecules. Chiral separations are accomplished by the addition of a chiral selector, which causes the different enantiomers of an analyte to focus at different positions along a microchannel or capillary. This new technique is demonstrated to provide high performance in a number of areas desirable for chiral separations including rapid separation optimization and method development, facile reversal of peak order (desirable for analysis of trace enantiomeric impurities), and high resolving power (comparable to capillary electrophoresis) in combination with greater than 1000-fold concentration enhancement enabling improved detection limits. In addition, chiral temperature gradient focusing allows for real-time monitoring of the interaction of chiral analyte molecules with chiral selectors that could potentially be applied to the study of other molecular interactions. Finally, unlike CE, which requires long channels or capillaries for high-resolution separations, separations of equivalent resolution can be performed with TGF in very short microchannels (mm); thus, TGF is inherently much more suited to miniaturization and integration into lab-on-a-chip-devices.

DNA hybridization assays using temperature gradient focusing and peptide nucleic acids.

Two types of DNA hybridization assays are demonstrated with temperature gradient focusing (TGF) and peptide nucleic acids (PNAs). In TGF, the application of a controlled temperature gradient along the length of a microchannel filled with an appropriate temperature-dependent buffer results in the formation of a gradient in both the electric field and electrophoretic velocity. Ionic species move in this gradient and concentrate at a unique point where the total velocity sums to zero. The first assay is a mixing assay in which PNA is allowed to flow through spatially focused DNA targets within a capillary. The second assay detects single base pair mutations (SBPM) by monitoring the fluorescence intensity of PNA/DNA duplexes as a function of temperature within the capillary. The SBPM analysis can be performed in less than 5 min with 100-fold more dilute analyte compared to conventional UV melting measurements.

Determination of triazine herbicides by capillary liquid chromatography with on-column focusing and temperature gradient

The combination of capillary liquid chromatography, temperature gradient and on-column focusing with large injection volumes offers a powerful tool for the determination of very low concentrations of triazines and their degradation products when very polar degradation products are present. A temperature gradient from subambient temperatures up to 70°C allows separation of eight triazines in 24 min. The focusing step makes it possible to increase 50 times the sample volume that can be injected into a capillary column. Detection limits (3×noise) are improved by a factor between 50 and 125 to 9 ng l −1 for hydroxyatrazine and 45 ng l −1 for atrazine. The chromatographic method, combined with solid phase extraction, was applied to the determination of eight triazines in ground water.