Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

Gong, Maojun; Wehmeyer, Kenneth R.; Limbach, Patrick A.; Heineman, William R.

doi:10.1002/elps.200600398

Cited by 21 publications

(16 citation statements)

References 32 publications

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“…R is the concentration ratio of the free (C f ) and the bound aptamers (C b ) in the incubated sample solution as expressed in Equation 2 (2) where A 1 , A 2 and A 3 are the areas of the complex, dissociated and free aptamers, respectively, as shown in Fig. 6; A front and A tail are the areas of complex fronting and freeaptamer tailing, respectively; t f and t c are the migration times of the free aptamer and complex, respectively; and α is an adjusting factor due to the dependence of the fluorescence intensity on the migration velocity of analytes [44]. The thrombin-aptamer complex dissociates during separation due to its rapid off-rate.…”

Section: K D Estimationmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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The use of traditional capillary electrophoresis (CE) to detect weak binding complexes is problematic due to the fast-off rate resulting in the dissociation of the complex during the separation process. Additionally, proteins involved in binding interactions often nonspecifically stick to the bare-silica capillary walls, which further complicates the binding analysis. Microchip CE allows flexibly positioning the detector along the separation channel and conveniently adjusting the separation length. A short separation length plus a high electric field enables rapid separations thus reducing both the dissociation of the complex and the amount of protein loss due to nonspecific adsorption during the separation process. Thrombin and a selective thrombinbinding aptamer were used to demonstrate the capability of microchip CE for the study of relatively weak binding systems that have inherent limitations when using the migration shift method or other CE methods. The rapid separation of the thrombin-aptamer complex from the free aptamer was achieved in less than 10 s on a single-cross glass microchip with a relatively short detection length (1.0 cm) and a high electric field (670 V/cm). The dissociation constant was determined to be 43 nM, consistent with reported results. In addition, aptamer probes were used for the quantitation of standard thrombin samples by constructing a calibration curve, which showed good linearity over two orders of magnitude with a limit of detection for thrombin of 5 nM at a 3-fold signal-to-noise ratio.

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Section: K D Estimationmentioning

confidence: 99%

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

et al. 2008

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“…The heating effect on the VEGF 165 -aptamer complex formation was studied using two sample solutions: one prepared with a heated aptamer stock solution (80 o C for 3 min), and the other with a non-heated aptamer. The results from frontal analysis [27] show that heating does not provide better complex signal than non-heating. Therefore, the aptamer was used without heating for all the other experiments.…”

Section: Condition Optimizationmentioning

confidence: 87%

“…The TG buffer with different additives was assessed to see its effect on the detection of the complex by frontal analysis using gated injection (Fig. 1b) [27]. A relatively large sample volume was pumped electrokinetically (4.5 kV between SR and BW for 3 s) into the separation channel followed immediately by the dispensing/separation step.…”

Section: Condition Optimizationmentioning

confidence: 99%

Detection of VEGF165 Using an Aptamer Affinity Probe in Microchip Capillary Electrophoresis

Gong¹,

Wehmeyer²,

Halsall³

et al. 2009

CPA

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Aptamers, like antibodies, have high molecular recognition specificity and selectivity. Coupled with microfluidic devices, apatamers and target-aptamer complexes can be rapidly separated while only a small sample volume is required. Trace protein detection using aptamer probes on a microchip platform was demonstrated using vascular endothelial growth factor 165 (VEGF 165 ) and a selective aptamer probe as a model system. The fluorescently labeled aptamer was separated from the VEGF 165 -aptamer complex in 10 s. The equilibrium dissociation constant of the complex was determined to be 8.0 nM using frontal analysis on the microchip. Two calibration curves were constructed with a detection limit of 1.0 nM VEGF 165 using pinched and gated injections. The blood plasma effects on the microchip electrophoretic separation and VEGF 165 -aptamer complex formation were investigated using rat blood plasma. Results demonstrate the power of microchip capillary electrophoresis in terms of assay speed, low reagent consumption and high separation efficiency. However, they also indicate the necessity of further improvements in the detection limit and/or the pretreatment of the plasma sample when measured by microchip CE.

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“…The same group recently developed a glass microchip with two-T injectors in CE/FA for studying the interaction between heparin and HSA with LIF detection [29]. Gong et al [30] used a glass microfluidic chip in a single-T structure to determine the dissociation constant of IgE-aptamer pair by modified gated injection with LIF detection.…”

Section: Design and Application Of Microchip Ce/fa For Free Bilirubinmentioning

confidence: 98%

“…The microchip CE provides the capability for the integration of sample pretreatment procedure prior to electrophoretic separation and detection. The FA format has recently been incorporated in microchip with a simple cross for sample introduction to study the interaction between simple and large molecule [27][28][29][30]. No work is found in the literature using microchip CE/FA for free bilirubin determination and study of its interaction with HSA.…”

Section: Introductionmentioning

confidence: 98%

Microchip capillary electrophoresis for frontal analysis of free bilirubin and study of its interaction with human serum albumin

Nie

Fung

2008

Electrophoresis

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To meet the need for bedside monitoring of free bilirubin for neonates under critical conditions, a microfluidic chip was fabricated and tested for its coupling with CE/frontal analysis (FA) to determine free bilirubin and study of its binding interaction with HSA, which regulated its concentration in plasma. The poly(methyl methacrylate) (PMMA) multichannel chip was fabricated by CO2 laser ablation and bonded with a fused-silica separation capillary for CE/FA separation with UV detection. The chip was designed to allow a complete assay of four electrophoretic runs using preconditioned channels to speed up the determination of free bilirubin and to deliver quick results for bedside monitoring. Under optimized conditions, the linear working range for free bilirubin was from 10 to 200 micromol with RSDs from 2.1 to 5.0% for n=3, and the LOD at 9 micromol for S/N=3. From a binding study between bilirubin and HSA under FA condition, the second binding constant for bilirubin-HSA was determined as 1.07x10(5) L/mol and the number of binding sites per HSA as 3.46. The results enabled the calculation of free bilirubin for jaundiced infants based on the clinically significant level of total bilirubin, producing a range of 118.3-119.4 micromol/L. The developed method is shown to meet the clinical requirement with additional margin of protection to detect the early rising level of free bilirubin prior to jaundice condition. The low-cost microchip CE/FA device is shown to produce quick results with high potential to deliver a suitable bed-side monitoring method for bilirubin management in neonates.

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Frontal analysis in microchip CE: A simple and accurate method for determination of protein–DNA dissociation constant

Cited by 21 publications

References 32 publications

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Protein‐aptamer binding studies using microchip affinity capillary electrophoresis

Detection of VEGF165 Using an Aptamer Affinity Probe in Microchip Capillary Electrophoresis

Microchip capillary electrophoresis for frontal analysis of free bilirubin and study of its interaction with human serum albumin

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