Isotachophoresis in Paper-Based Microfluidic Devices

Abstract

Paper substrates have been widely used to construct microfluidic devices for use in rapid point of care (POC) diagnostic testing. Use of paper materials to construct these tests has grown from their success in lateral flow (LF) immunoassay diagnostics, such as home pregnancy test, malaria, flu (H1N1), and HIV tests. Paper based microfluidic devices are robust, low-cost, and relatively simple to operate, compared to channel microfluidic devices, which is perhaps their greatest advantage and the reason they have reached a high level of commercial success. However, the growing demand for improved detection limits is challenging the current format of paper based diagnostic tests including LF assays. Moreover, paper devices may not be well suited for integrated sample preparation, such as sample extraction and preconcentration, that is required in complex matrices when samples with low traces of target molecules are available. In this dissertation isotachophoresis (ITP) is used to preconcentrate and separate analytes on paper substrate in the goal to improve detection limits of paper based microfluidic devices. ITP is an effective and robust electrokinetic technique which has the potential to simultaneously preconcentrate and separate target analytes on the paper substrate. In the first phase of this work, we present ITP preconcentration of a fluorescence dye on nitrocellulose membrane. ITP has the potential to be performed on nitrocellulose membrane and increase concentration of target molecules. It also has the capacity to extract large amount of target analytes from a complex matrix where the analyte of interest is dilute but the sample volume can be large. Our findings show that ITP on nitrocellulose is capable of up to 900 fold increase in initial sample concentration and up to 60% extraction from 100 µL samples and more than 80% extraction from smaller sample volumes by using finite sample injection. We show that ITP on nitrocellulose membrane can be powered and run several times by a small button battery suggesting that it could be integrated to a portable POC diagnostic device. These results highlight the potential of ITP to enhance detection limits of paper based LF assays. In the second part, we use ITP to improve detection limits of LF assays. Binding reaction in LF assays, which results in detection of a target, can be modeled as a second-order surface reaction with on- and off- rate constants. At low target concentration this reaction is kinetically limited, meaning that the binding reaction can take several hours to result in a detectable signal. We use ITP to preconcentrate target molecules in solution and transport them to a thin band of immobilized probe resulting in an increase in kinetic reaction rates of the target and the probe. We show that ITP-enhanced LF (ITP-LF) is able to improve limit of detection (LOD) of conventional LF assays by 160-fold, at a 5 minutes time scale. ITP-LF shows up to 30% capturing of the target from 100 µL of the sample, while conventional LF captures less than 1% of the target. High capturing efficiency provided by ITP-LF assay is valuable especially when trace amount of target is available in a large volume of the sample, e.g. blood, saliva. We also show that ITP is able to separate and concentrate target analyte from a complex sample and transport them on the paper substrate. This is one step toward integration of complex samples and assays, e.g. nucleic acid purification, on paper-based microfluidic devices.