Demonstration and Characterization of Biomolecular Enrichment on Microfluidic Aptamer-Functionalized Surfaces

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.

Figure 1

Principle of aptamer-based microfluidic enrichment. (a) A mixture of target and impurities introduced to a specific aptamer on a surface (for example, microbeads) at a certain temperature. (b) Rinsing out impurities and purifying the analyte (c) Enrichment of analytes via addition of subsequent dilute sample, or continuous infusion after a short time Δt. (d) Release of the target molecule at a suitably different temperature.

Figure 2

(a) Schematic of the microchip. (b) Magnified view of the aptamer microchamber highlighting the weir structure. Microbeads are trapped provided the height of the weir is smaller than 0.5× the nominal bead diameter. (c) Micrograph of a packaged device.

Figure 3

Control experiments. A 11.7 μM sample of TO-AMP was injected into the microchamber containing three SPE media: 1) non-functional beads (“aptamer neutral”); 2) beads coated with nonspecific aptamer (GTP-aptamer, “aptamer negative”); 3) AMP-specific aptamer coated beads (“aptamer positive”). Insets show fluorescence micrographs of the chamber corresponding to each control condition. Experiments were performed in triplicate to determine a standard error, which procedurally was similar for all subsequent figures containing error bars.

Figure 4

Time course of resolved fluorescence measurements for the extraction of TO-AMP by its aptamer. Lines represent a nonlinear least squares fit to an exponential association model.

Figure 5

Temperature induced dissociation (i.e., isocratic elution) of TO-AMP from the aptamer using the integrated heating elements on the microchip. The release profiles suggest the microchip is capable of rapid (τ_diss = 1.31 s, average) and thorough release and elution of TO-AMP as compared to room temperature dissociation (τ_diss = 16.5 s when considering the 6.5 μM case).

Figure 6

Enrichment by continuous infusion of an impure and dilute sample of TO-AMP (10 nM). A solution of TO-AMP is spiked with a model impurity (TMR-AVP) at a concentration ratio of 100:1, which is a common neurophysical hormone present alongside AMP physiologically. The red, green and blue channel for each fluorescence data point is extracted and plotted to measure the efficiency of TO-AMP purification by the aptamer.

Figure 7

Equilibrium binding fluorescence response of TO-AMP within the linear binding range. Inset is a magnification of the low concentration range (0–7000 nM).

Figure 8

Enrichment by discrete infusion of dilute samples of TO-AMP. Lines represent a least squares fit of data to Eq. (4). As expected, the change in concentration between successive concentrations approaches zero as the number of experimental infusions increase, exhibiting a nonlinear trend.

Figure 9

Enrichment by continuous infusion of dilute samples of TO-AMP. In spite of the non-discrete method in which this experiment is performed, Eq. (5) of our model can still be used to predict the final enrichment factor for each tested TO-AMP concentration.