An aptamer-based microfluidic device for thermally controlled affinity extraction

Microfluidics and Nanofluidics (Impact Factor: 2.53). 04/2008; 6(4):479-487. DOI: 10.1007/s10404-008-0322-4


We present a microfluidic device for specific extraction and thermally activated release of analytes using nucleic acid aptamers.
The device primarily consists of a microchamber that is packed with aptamer-functionalized microbeads as a stationary phase,
and integrated with a micro heater and temperature sensor. We demonstrate the device operation by performing the extraction
of a metabolic analyte, adenosine monophosphate coupled with thiazole orange (TO-AMP), with high selectivity to an RNA aptamer.
Controlled release of TO-AMP from the aptamer surface is then conducted at low temperatures using on-chip thermal activation.
This allows isocratic analyte elution, which eliminates the use of potentially harsh reagents, and enables efficient regeneration
of the aptamer surfaces when device reusability is desired.

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    • "The chip consists of an aptamer microchamber packed with microbeads whose surfaces are functionalized with the aptamer. Compared with the earlier design [19], the microchamber volume is reduced to decrease detection signal nonuniformity (significant with larger sized microchambers), and to ensure that the sample is exposed to all available aptamer binding sites. The microbeads are introduced from a dedicated bead inlet and retained in the chamber using a dam-like structure termed a weir (Fig. 2b), while samples and buffer are introduced via the sample inlet and outlet. "
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    ABSTRACT: This paper demonstrates and systematically characterizes the enrichment of biomolecular compounds using aptamer-functionalized surfaces within a microfluidic device. The device consists of a microchamber packed with aptamer-functionalized microbeads and integrated with a microheater and temperature sensor to enable thermally controlled binding and release of biomolecules by the aptamer. We first present an equilibrium binding-based analytical model to understand the enrichment process. The characteristics of the aptamer-analyte binding and enrichment are then experimentally studied, using adenosine monophosphate (AMP) and a specific RNA aptamer as a model system. The temporal process of AMP binding to the aptamer is found to be primarily determined by the aptamer-AMP binding kinetics. The temporal process of aptamer-AMP dissociation at varying temperatures is also obtained and observed to occur relatively rapidly (< 2 s). The specificity of the enrichment is next confirmed by performing selective enrichment of AMP from a sample containing biomolecular impurities. Finally, we investigate the enrichment of AMP by either discrete or continuous introduction of a dilute sample into the microchamber, demonstrating enrichment factors ranging from 566 to 686×, which agree with predictions of the analytical model.
    Preview · Article · Jul 2011 · Sensors and Actuators B Chemical
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    • "This paper presents a microfluidic aptamer-based biosensor for cocaine that uses a cocaine-specific DNA aptamer modified with a fluorophore. Based on a device architecture designed for highly specific solid-phase extraction [17] [18], this sensor is capable of lowcost quantitative cocaine detection in a highly specific, signal-on, and label-free manner. We demonstrate that the device is capable of specifically detecting cocaine at concentrations as low as 10 pM, with a linear response over four orders of magnitude. "
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    ABSTRACT: We present a microfluidic aptamer-based biosensor for specific cocaine detection. The device consists primarily of a microchamber packed with aptamer-functionalized microbeads that act as a sensing surface, integrated with an on-chip heater and temperature sensor. The sensor employs a Förster resonance energy transfer (FRET) system in which a fluorophore-quencher pair of carboxyfluorescein and Dabcyl generates a signal-on response to cocaine. We demonstrate device operation by successfully detecting cocaine with a four orders of magnitude linear response in micromolar to nanomolar concentrations. The detection limit of the device is further lowered to 10 pM by concentration of a highly diluted cocaine sample, which compares well with the most sensitive detection techniques currently available. The temperature-dependent binding of aptamer–analyte complexes is then used to effect thermal release of cocaine from the sensing surface. It is found that a temperature of 37 °C can fully regenerate the sensor in pure buffer. Furthermore, testing indicated that sensor response is consistent after repeated regeneration. These results demonstrate that aptamer-based sensing on a microfluidic platform has the potential to enable low-cost, rapid, and highly specific detection of cocaine in practical applications.
    Preview · Article · Apr 2011 · Sensors and Actuators A Physical
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    • "The signal gain achieved in Fig. 6(b) is merely the apparent signal enhancement, since the potential for even larger enrichment factors and higher signal gain is possible with our microchip [34]. It is also significant to mention that although the repeatability of our experiments is not explicitly gleaned from the presented data (due to the limits of presenting spectroscopic data), the microchip was easily regenerated (using thermal stimulation of the aptamer-functionalized beads) to allow reuse and repeated functionality [17]. "
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    ABSTRACT: We present an innovative microfluidic system that accomplishes specific capture, enrichment, and isocratic elution of biomolecular analytes with coupling to label-free mass spectrometric detection. Analytes in a liquid phase are specifically captured and enriched via their affinity binding to aptamers, which are immobilized on microbeads packed inside a microchamber. Exploiting thermally induced reversible disruption of aptamer-analyte binding via on-chip temperature control with an integrated heater and temperature sensor, the captured analytes are released into the liquid phase and then isocratically eluted and transferred via a microfluidic flow gate for detection by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The utility of the device is demonstrated using adenosine monophosphate (AMP) as a model analyte. Experimental results indicate that the device is capable of purifying and enriching the analyte from a sample mixed with nonspecific analytes and contaminated with salts. In addition, thermally induced analyte release is performed at modest temperatures (45degC), and mass spectra obtained from MALDI-MS demonstrate successful detection of AMP at concentrations as low as 10 nM following enrichment by consecutive infusion of a diluted sample.
    Preview · Article · Jan 2010 · Journal of Microelectromechanical Systems
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