1,000-fold sample focusing on paper-based microfluidic devices
Tally Rosenfeld and Moran Bercovici
We have developed the fabrication process of a novel paper-based analytical device (μPAD) for isotachophoretic sample focusing. Using this device we demonstrated the processing of 30 μL of sample achieving 1,000-fold increase of peak concentration in 6 min.
Guided by a simple heat transfer model, we further developed wax-printing fabrication to enable the creation of shallow channels which are critical in providing sufficient dissipation of joule heat, and thus enable the use of high electric fields and short analysis time. This results in a device that is self-contained on a simple piece of paper and does not require any specialized enclosures or cooling devices to combat evaporation of high temperatures.
We have provided an analytical model for isotachophoretic sample accumulation in porous media, introduce a simple figure of merit for evaluating and comparing the efficiency of such devices, and present experimental validation in both paper and glass channels.
Paper-based microfluidic devices
Microfluidic paper-based analytical devices (μPADs) are a new class of point-of-care diagnostic devices that are inexpensive, durable and simple to use. Such devices have now been applied for detection of glucose, heavy metals, total proteins, and ELISA. The most common use of μPADs to date is in lateral flow strips, which can be found in applications ranging from malaria detection in developing countries, to home pregnancy tests. However, these examples represent few cases in which the clinically relevant concentration of the target protein (here hCG and HRP II) are compatible with the sensitivity of flow strips. Despite well identified biomarkers, many diagnostic needs cannot be met by the current sensitivity of such tests. Low-cost and rapid assays capable of accurately and sensitively detecting disease at the point-of-care would have a significant impact on global health, enabling access to advanced molecular diagnostics even in under-resourced and rural areas. Our goal is to enhance the sensitivity of such paper-based devices by incorporating (on paper) electrokinetic techniques such as Isotachophoresis (ITP).
Fabrication of shallow channel μPADs
Our fabrication technique is based on wax printing, in which wax is directly printed onto cellulose. However, for compatibility with electrokinetic assays, we further developed the technique to allow the fabrication of channels significantly shallower than the original thickness of the paper (~50 µm). Such shallow channels are critical in providing sufficient dissipation of joule heat and thus enable the use of high electric fields and short analysis time.
Figure 1: (a) Schematic illustration of the multistep fabrication process. We print the channel side wall template (black regions) on one side and a thick wax layer (orange region) on the other side of the paper. The channel is designed to be 2.5 mm wide and is connected to 6.5 mm radius round reservoirs. We then pass the paper through a temperature-controlled laminator which melts the wax and allows it to penetrate to the desired depth. Finally, to prevent evaporation, we cover the channel with transparent tape, while both reservoirs are kept open to the atmosphere. (b) 3D illustration of the resulting structure. On one end of the channel (TE reservoir) we print a hydrophobic wax barrier, which stops the flow of the LE and serves as a repeatable starting point for ITP. (c) Raw fluorescence images of the paper cross section showing the effect of lamination temperature on the penetration of wax into the paper, resulting in control of the channel depth. (d) An image of our fabricated paper-based microfluidic device including dimensions.
Figure 2:Demonstration of the use of our fabricated μPAD for ITP focusing. (a) We place electrodes in each of the reservoirs, and add the LE to the right reservoir (b) The channel is filled with the LE buffer by capillary action (c) After ~10 min, when the LE front stops at the barrier, we add a TE-sample mixture to the left reservoir (d) Contact is formed between the TE and LE buffers, and ITP automatically initiates. (e) Raw fluorescence image of ITP focusing of 1 μM fluorescein on a filter paper, imaged by a consumer-grade camera (f) Typical fluorescence image of ITP focusing imaged under a microscope.
Video 1: Demonstration of the use of our fabricated μPAD for ITP focusing.
1,000-fold sample focusing on μPAD
Figure 3:<\em>Experimental results showing continuous ITP focusing of a fluorescent dye on a filter paper channel. We injected 10 nM DyLight650 into the TE, and measured fluorescence intensities during ITP, at fixed distances from the TE reservoir (stations are printed as 1-8 in Figure 2). (a) Width averaged concentration profiles registered at each station. While the ITP front is significantly more dispersed compared to standard glass microchannels, focusing is nevertheless clearly evident and the ITP plug is well contained and steadily electromigrates along the paper channel. (b) Total accumulated sample at each station (calculated using values above 10% of the peak value threshold), showing continuous accumulation of sample at the interface. The solid line represents ITP theory for constant voltage (c) Raw intensity images corresponding to each station.
Figure 4: Experimental measurements of focusing ratio achieved in paper ITP. (a) Raw fluorescence image of the focusing zone. The analyte’s maximum concentration is denoted by Cpeak. (b) Measurements of area-averaged and peak concentrations, based on four repeats (various symbols), showing a 200-fold and a 1,000-fold concentration increase, respectively, achieved in less than 6 min. The peak concentration is important for detection and imaging applications, while the average concentration is important in applications where ITP is used to accelerate the reaction between co-focusing species which determines the rate of reaction.