Enzyme reactions in nanoporous, picoliter volume containers

Abstract

Advancements in nanoscale fabrication allow creation of small-volume reaction containers that can facilitate the screening and characterization of enzymes. A porous, ∼19 pL volume vessel has been used in this work to carry out enzyme reactions under varying substrate concentrations. Assessment of small-molecule and green fluorescent protein diffusion from the vessels indicates that pore sizes on the order of 10 nm can be obtained, allowing capture of proteins and diffusive exchange of small molecules. Glucose oxidase and horseradish peroxidase can be contained in these structures and diffusively fed with a solution containing glucose and the fluorogenic substrate amplex red through the engineered nanoscale pore structure. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Kinetic characteristics of the enzyme (K(m) and V(max)) were evaluated and compared with results from conventional scale reactions. These picoliter, nanoporous containers can facilitate quick determination of enzyme kinetics in microfluidic systems without the requirement of surface tethering and can be used for applications in drug discovery, clinical diagnostics, and high-throughput screening.

Figure 1

A nanoporous, picoliter volume device platform for enzyme reactions. (a) SEM micrograph of a microfluidic device with an array of 18 reaction containers. The inset SEM micrographs show a single reaction container and a part of the container wall showing the nanoscale slits. After PECVD, the slits have a limiting aperture of ~10 nm. (b) A fluorescent micrograph of an array of enzyme reactions being carried out simultaneously. Enzyme concentrations of either 0.125 U/mL to 0.25 U/mL of glucose oxidase (GOX) and horseradish peroxidase (HRP) are contained in the devices and 10 mM glucose is flowed in the channel. (c) Time dependent trace of the normalized fluorescence intensity in a reaction container (0.25 U/mL GOX and 0.25 U/mL HRP) as flow is alternated between buffer with and without 10 mM glucose. Error bars representing +/- one standard deviation are shown at 50 seconds intervals for clarity.

Figure 2

Experimental diffusion data and diffusion model for devices that have undergone 4, 5, 6 and 7 minutes of PECVD. Solid data points correspond to the relative, average fluorescein intensity in the device and result from three diffusion experiments (3 individual devices on 3 separate chips). The solid lines correspond to the calculated fluorescein concentration using a Lumped Capacitance model and a pore size of either 9, 13, 25 or 35 nm. An increased duration of PECVD decreases the observed pore size of the device. For comparison, the open circles correspond to GFP diffusion data in a device coated with 7 minutes of PECVD, and the dashed line corresponds to a model of GFP diffusion using a 5 nm pore.

Figure 3

Fluorescently measured single enzyme reaction in picoliter volume reactors. Devices that have undergone 7 min PECVD were filled with 0.25 U/mL horseradish peroxidase (HRP) and exposed to 2.5μM hydrogen peroxide (H₂O₂) using a constant flow rate of 10 μL/hr. Representative fluorescent micrographs of the cell mimic devices taken a different time points are shown. Error bars representing +/- one standard deviation of 3 individual devices on 3 separate chips are shown at 20 seconds intervals for clarity.

Figure 4

Substrate dependent fluorescence response for coupled enzyme reactions in picoliter volume reactors. As glucose concentration increases from 10 μM to 100 mM, the rate of product formation, as measured by resorufin fluorescence, increases. Each data point represents the resorufin concentration, based on the observed fluorescence intensity, from three coupled enzyme reaction experiments (3 individual devices on 3 separate chips). Error bars represent +/- one standard deviation and are shown at 60-second intervals for clarity.

Figure 5

Nonlinear regression fitting of Michealis-Menten plots of coupled enzyme reactions in picoliter volume reaction device (ν) and plate reader (λ). The K_m and V_max for the reaction device were found to be 1.65 ± 0.17 mM and 67 ± 1.5 μM min^-1 respectively. For comparison, the K_m and V_max for the coupled enzyme reactions carried out in a plate reader (100 μL volume) were found to be 0.75 ± 0.04 mM and 25 ± 0.3 μM min^-1 respectively. The inset expands the low glucose concentration region.