Isotachophoretic Preconcenetration on Paper-Based Microfluidic

May 13, 2014 - Paper based microfluidic devices are robust and relatively simple to ... and the reason they have reached a high level of commercial su...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/ac

Isotachophoretic Preconcenetration on Paper-Based Microfluidic Devices Babak Y. Moghadam, Kelly T. Connelly, and Jonathan D. Posner* Department of Mechanical Engineering, University of Washington, Seattle, Washington 98195, United States S Supporting Information *

ABSTRACT: Paper substrates have been widely used to construct point-of-care lateral flow immunoassay (LFIA) diagnostic devices. Paper based microfluidic devices are robust 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, paper devices may not be well suited for integrated sample preparation, such as sample extraction and preconcentration, which is required in complex samples with low analyte concentrations. In this study, we investigate integration of isotachophoresis (ITP), an electrokinetic preconcentration and extraction technique, onto nitrocellulose-based paper microfluidic devices with the goal to improve the limit of detection of LFIA. ITP has been largely used in traditional capillary based microfluidic devices as a pretreatment method to preconcentrate and separate a variety of ionic compounds. Our findings show that ITP on nitrocellulose is capable of up to a 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. Paper based ITP is challenged by Joule heating and evaporation because it is open to the environment. We achieved high preconcentration by mitigating evaporation induced dispersion using novel cross-shaped device structures that keep the paper hydrated. We show that ITP on the nitrocellulose membrane can be powered and run several times by a small button battery suggesting that it could be integrated to a portable point-of-care diagnostic device. These results highlight the potential of ITP to increase the sensitivity of paper based LFIA under conditions where small analyte concentrations are present in complex biological samples.

I

distinct analyte zones each at a locally uniform concentration.10,11 At trace level concentrations, sample ions rarely form a plateau zone and operate in peak mode ITP where sample ions accumulate in a concentrated sample zone between LE and TE, which we refer to as an ITP plug. Various articles have described the physics of peak mode ITP using numerical, analytical, and experimental methods. Khurana and Santiago theoretically and experimentally studied sample zone dynamics in peak mode ITP in a glass capillary. They varied experimental parameters governing the sample zone dynamic independently, e.g., electrolytes concentration and applied current, to validate their analytical models and to provide a guide to experimental design and optimization of practical ITP assays.12 They reported that, in contrast to plateau mode ITP, there is an optimum LE concentration in peak mode ITP at which the highest preconcentration can be reached. They also showed that higher current densities and lower TE concentrations result in higher preconcentration ratios. Garcia-Schwarz et al. developed theoretical and numerical models to account for sample dispersion in peak mode ITP and showed nonuniform

sotachophoresis (ITP) is a nonlinear electrophoretic technique used to preconcentrate and separate a variety of ionic compounds, ranging from small metallic ions1 to large biomolecules such as proteins and nucleic acids. ITP is an effective electrophoretic preconcentration technique, with the potential of up to 1 million fold preconcentration.2−4 In ITP, sample ions focus between leading (LE) and trailing electrolytes (TE) which have co-ions with respectively higher and lower effective electrophoretic mobilities than the sample ions. When a constant voltage or current is applied across the channel, sample ions accumulate and preconcentrate by electrophoresis into a number of contiguous zones between LE and TE zones, arranged in the order of their mobilities. Each zone has uniform characteristic concentration governed by electrophoresis conservation laws, namely the Kohlrausch regulating function5,6 (for fully ionized species) or more generally the Jovin−Alberty function7,8 (for weak analytes), which calculate the adjusted concentrations of species in each zone. The adjusted concentration is the concentration each species obtains by electromigration into the zones previously occupied by species with another composition. Depending on the initial concentration of the target sample, it can be focused (concentrated) in plateau mode or peak mode ITP.9 High initial sample concentrations and sufficient focusing time result in plateau mode ITP, which is characterized by © 2014 American Chemical Society

Received: February 12, 2014 Accepted: May 13, 2014 Published: May 13, 2014 5829

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry

Article

axial counter electro-osmotic flow (EOF) results in sample dispersion. In addition, samples with mobility values near those of the TE or LE show greater diffusion into the TE or LE, respectively.13 They showed that advective dispersion caused by the nonuniform counter EOF when coupled with other sources of dispersion can drastically reduce the ITP preconcentration ratio. Their models allow for fast and accurate prediction of dispersed sample distributions in ITP based on known parameters including species mobilities, electro-osmotic (EO) mobility, applied current density, and channel dimensions. Recently, cellulose based membranes have been used as a substrate to construct microfluidic devices for use in rapid diagnostic tests.14 Use of paper (or membranes) in microfluidic devices has grown from their success in immunochromatographic (also called lateral flow or dipstick test) point of care diagnostics,15,16 such as a home pregnancy test strip. Typically, these tests are based on a strip of membrane immobilized with a captured antibody specific to an antigen of interest.17 Paper based point of care diagnostic devices promise to be inexpensive, user-friendly, rapid, portable, disposable, and readable by eye with visible signals. Many of these paper based devices are thus focused on applications for use at home or in developing countries.18 The growing demand for increased analytical sensitivity is challenging the current format of lateral flow immunoassay (LFIA) tests.19 Target biomolecules present at low concentrations require novel detection, amplification, and sample pretreatment methods to improve their limits of detection (LOD). Many attempts have been made to improve LOD in LFIA, including labeling the bioreceptors with colloidal particles,20 chemical and colorimetric signal amplification,21 enzymatic signal amplification,22 and using multistep sample processing in two-dimensional paper networks.23 However, these methods require additional instrumentation, sophisticated chemical processes, and a significant increase in the testing time. Fernández-Sánchez et al. reported one of the lowest detection limits for traditional LFIA on a polyethylene-based membrane at 1 μg/L.24 They sandwiched prostate specific antigen (PSA) between anti-PSA antibodies immobilized on the strip and a colloidal gold anti-PSA antibody tracer. They were able to achieve low LOD by introducing an extra washing step to the assay in order to reduce the background noise, but this washing step requires further manipulation by the user after sample addition and may negatively influence the reproducibility and reliability of the device. Electrokinetic techniques have been used for 50 years on porous thin layers and membranes to separate charged molecules of small and intermediate size, e.g., paper chromatography and paper electrophoresis.25−27 These studies use electrophoretic transport to separate and analyze chemical compounds. In a recent study by Oyama et al., they developed a quantitative immunoassay point of care (POC) chip with a glass porous fiber sheet, which has a fabric structure similar to cellulose paper.28 They used EOF to drive fluorescently labeled target molecules to the capture zone and compared the sensitivity of their device to conventional capillary flow based LFIA. Their method lacked specificity to particular analyte molecules in a complex matrix, required a complicated fabrication procedure and a minimum test time of 20 min, and did not achieve a LOD which is consistent with conventional lateral flow tests. We hypothesize that peak mode ITP can be a promising rapid and portable technique to improve LOD of paper-based LFIA by extracting specific

molecules in a complex matrix and by increasing their concentration at the location of the test zone. There have been a few previous attempts to integrate ITP on membranes, mostly with the goal of separating ions. In the 1970s, Taglia and Lederer showed the feasibility of integrating plateau mode ITP on cellulose filter paper strips to separate a mixture of inorganic ions.29 They were able to qualitatively show separation of different ions on the filter paper, but excessive heating of the paper limited them to voltages lower than 400 V and ionic strengths lower than 100 mM, and each separation experiment took 3 h to complete. Abelev and Karamova conducted the first studies to qualitatively separate and concentrate proteins using plateau mode ITP on membranes during the 1980s. They conducted their experiments at relatively low voltages, e.g. 200 V, which took 4 h to be completed, and the cellulose acetate membranes they used have low protein binding capacity and thus have limited application for LFIA.30 In this paper, we study the use of peak mode ITP on nitrocellulose membranes with the goal of improving LOD of paper-based LFIA. ITP on nitrocellulose has the potential to target specific molecules in a complex matrix and increase their concentration at the test zone. It also has the capacity to operate with a relatively large volume of sample, up to 100 μL, to extract a large fraction of the sample where the analyte of interest is dilute but the sample volume can be large. We chose a nitrocellulose membrane to conduct ITP experiments since it has been widely applied to LFIA tests and paper-based microfluidic devices.31 The structure and porosity of nitrocellulose membranes are highly controllable, have a high contrast background for colorimetric assays, are inexpensive, and have high binding capacity for biomolecules.32 We perform several studies aimed at designing and optimizing ITP conditions and chemistry. We provide an extensive quantitative analysis of the ITP preconcentration by reporting the stacking ratio and amount of sample accumulated in the sample zone. Stacking ratio is defined as the ratio of the sample concentration in the plug to its initial concentration in the TE reservoir. We present a cross-shaped membrane geometry that results in larger ITP stacking ratios by mitigating sample dispersion induced by evaporation from the membrane free surface and Joule heating. Finally, in a step toward miniaturization and commercialization of ITP based paper microfluidic devices, we show that a portable, watch-battery powered electronic circuit can be used to perform ITP on nitrocellulose membranes with performance consistent with a regulated benchtop power supply.

■

EXPERIMENTAL SETUP AND PROTOCOLS Materials. We performed a series of anionic ITP experiments to show the capability of this technique in preconcentration of a target sample on a nitrocellulose membrane. We chose Alexa Fluor 488 (AF488) succinimidyl ester (Molecular Probes, Eugene, OR) as the sample and designed the chemistry of the electrolytes based on that.3,13 We use AF488 because of its extensive use in conventional ITP assays and its exceptional optical stability in a wide range of pHs.3,4,12 The use of this dye also removes the complexities introduced by adsorption to the membrane surface that will be present when separating biomolecules. The leading electrolyte (LE) consisted of 40 mM HCL, fast moving ions, in 80 mM Tris buffer. The trailing electrolyte (TE) was 10 mM HEPES, slow moving ions, in 20 mM Tris buffer, mixed with 50 nM of sample. We chose the 5830

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry

Article

buffering counterions species and the pH of the LE and TE electrolytes in such a way that maximal differences in effective mobilities can be obtained. Effective mobility is a function of the degree of dissociation of analytes which is typically a function of their dissociation constants (pKa), local pH, and local ionic strength.33 We selected Tris as the counterion and buffering agent since it is positively charged, and it has strong buffering capacity at pH = 8 due to its pKa value, which does not exceed more than 1 pH unit from that of the electrolytes.34 Total ionic strength, i.e. conductivity, of the TE was chosen to be lower than that of the LE in order to increase the flow of sample ions into the ITP plug, as will be discussed in detail in the Results and Discussions sections. For a detailed discussion on the choice of electrolyte system in ITP, refer to the works of Bagha et al. and Everaerts et al.10,33 We added 3% polyvinylpyrrolidone (PVP) to the LE to suppress counter EOF.35 All the chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless mentioned otherwise. All aqueous samples were prepared using water ultrapurified with a Milli-Q Advantage A10 system (Millipore Corp., Billerica, MA). Instrumentation. We performed quantitative fluorescence imaging to visualize anionic dye focused by steady-state ITP experiments using a Nikon AZ100 microscope equipped with 0.5x (NA 0.05) and 5x (NA 0.5) magnification objectives (Nikon Corporation, Tokyo, Japan), an epifluorescence filter cube (488 nm excitation, 518 nm emission, Omega Optics, Brattleboro, VT), and a 16-bit, cooled CCD camera (Cascade 512B, Photometrics, Tucson, AZ). Figure 1A shows a schematic of our ITP experimental setup. A high voltage power supply (HSV488 6000D LabSmith Inc., Livermore, VT) applied a constant electric current. Digital processing and analysis of the data, images, and movies was performed by a custom code written in MATLAB (MathWorks Inc., Natick, MA). We fabricated our paper devices from a backed nitrocellulose membrane (HF-135, Millipore, Billerica, MA) cut by a CO2 laser (Universal Laser Systems, Scottsdale, AZ). Figure 1B shows the straight and cross-shaped structures we used. Structures used in this work have a constant width (W) of 3.5 mm and varying lengths (L) of 35, 40, and 45 mm. We use the cross-shaped designs to mitigate membrane drying and the resultant decrease in preconcentration due to dispersion. We chose the width of the cross wings to be half of the width of the membrane to minimize diffusion of sample ions into the wings, and their location to be at L/3 from the TE reservoir close to the location where drying usually starts. Laser cut acrylic sample holders, with four 100 μL reservoirs, held the paper devices on the microscope stage (Figure 1A). We folded the west and east ends of the membranes and dipped them into the TE and LE electrolyte reservoir, respectively. For the cross-shaped strips, we folded the north and south wings and dipped them into the reservoirs filled with DI water to provide moisture by capillary action to the membrane during the ITP experiments. Platinum wires, dipped in the acrylic reservoirs, conveyed the applied voltage to the paper devices as shown in Figure 1A. ITP Protocol. We rinsed all the reservoirs and platinum wires with DI water several times before starting the experiments to reduce any contamination. We wet the nitrocellulose side of the membrane by adding a few drops of LE from the LE side so that three-fourths of the paper is wet. This wetting procedure reduces the time for the LE to wet the membrane by the capillary flow. We then placed the membrane on the chip with the backing side facing up and the folded ends

Figure 1. Schematics of (A) the ITP experimental setup. TE reservoir (on left) and LE reservoir (on right) etched on the acrylic chip. Direction of the anionic ITP is from the negative electrode on the TE side to the positive electrode on the LE side. (B) Devices used to conduct ITP experiments. Simple straight membrane 3.5 mm in width and varying in length and a cross-shaped membrane for minimizing sample evaporation. Width of the wings was chosen to be half the width of the membrane to minimize the ITP flow disturbance by the cross-flow.

dipped in the west (negative) and east (positive) reservoirs. The east reservoir was already filled with LE, whereas 100 μL of the TE mixed with the sample was added to the west reservoir after the membrane was placed on the chip. The membrane wicks the solution as the TE reservoir is filled with the electrolyte. After establishing an interface between the TE and LE, constant electric current is applied across the two reservoirs. AF488 ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE/TE interface. We acquired images of the concentrated sample zone (ITP plug) as it generates and travels from the TE reservoir toward the LE reservoir in a field of view that covers 27 mm of the membrane upstream of the LE reservoir (dashed lines in Figure 1). We performed calibration experiments before and after running each set of experiments. We measured fluorescence intensity of the membrane fully wetted by the homogeneous concentration of dye at three different molar concentrations of 10 μM, 25 μM, and 50 μM. In the ITP experiments, we calculate the sample concentration, CSample, as 5831

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry CSample =

Cdye Idye − Iback

Article

(ISample − Iback )

(1)

where ISample is the fluorescence intensity of the stacked sample, Idye is the signal intensity corresponding to a known concentration of highly concentrated dye, Cdye, and Iback is the background intensity measured when the membrane is fully wetted by the buffer, i.e., zero dye concentration. In practice, Cdye/Idye −Iback is the slope of the linear regression fit to the calibration points using the least-squares method. Captured images from the ITP plug were width averaged (transverse, y direction) and fit with a Gaussian distribution.4 Khurana and Santiago showed that in peak mode ITP, the concentration profile of the sample zone is approximately Gaussian rather than plateau shaped,36 which is governed by a local Taylor−Aris-type dispersion. We found good agreement between the experimentally measured concentration and Gaussian distributions. The Gaussian fits are used to determine the reported maximum intensity of the sample in the plug. An example of sample zone fluorescence intensity distribution and Gaussian fit is provided in the Supporting Information (SI).

■

RESULTS AND DISCUSSIONS Concentration and accumulated moles of the sample in the sample zone are critical figures of merit in determining ITP signal strength and sensitivity and are governed by several parameters including the chemistry of the electrolytes, applied electric field, and different sources of sample dispersion.12 In order to achieve higher sample stacking ratios, we determined how varying these parameters along with the length of the device affect the ITP preconcentration on nitrocellulose membrane. Plug Formation and Effect of Counter EOF. Figure 2 shows normalized spatiotemporal maps of the ITP plug as the axial location on the x-axis changes with time on the y-axis. Figure 2A and B show the plug migration and concentration without and with 3% PVP in the LE, respectively. These plots are generated by averaging fluorescence intensity of the plug in the transverse direction for a single ITP experiment. Net migration velocity of the ITP plug as it travels from the TE side (on left) toward the LE side (on right) can be found by calculating the slope of the line in these maps, v = dx/dt. These plots show how a highly dispersed cloud of sample ions appears on the membrane at the boundary of LE/TE after applying the electric field at t = 0. As the concentration of the cloud increases, a plug with sharp boundaries forms and travels downstream toward the LE reservoir. We have provided example videos of the ITP plug formation and migration in the SI. The plug migration is due to a combination of electromigration and counter electro-osmotic flow. Without PVP, the plug velocity slows and reverses direction toward the TE reservoir because the opposing EOF velocity becomes larger than the plug electromigration velocity, as shown in the inset of Figure 2A. By adding PVP to the LE, as shown in Figure 2B, the velocity of the plug increases significantly, indicating that the EOF is being suppressed. Axial counter EOF is a source of convective dispersion of the sample in isotachophoretic focusing which has a significant impact on the ITP preconcentration.13 Our detailed quantitative analysis presented in the SI show that the addition of 3% PVP to the LE sufficiently decreases EOF-induced sample dispersion, resulting

Figure 2. Spatiotemporal maps of the sample zone as it travels downstream of the TE reservoir for a single ITP experiment for (A) 0% and (B) 3% PVP in the LE. The inset of A shows that at longer times the traveling direction of the ITP zone reverses due to counter EOF. Adding PVP to the LE suppresses the counter EOF, resulting in a higher stacking ratio and reduction of time needed for each experiment. Here, the TE is 20 mM HEPES and 40 mM Tris mixed with 50 nM AF488. LE is 40 mM HCl and 80 mM Tris. Applied current is 500 μA, and length of the membrane is 40 mm.

in higher stacking ratios and ITP plug velocity. Therefore, we use a 3% PVP concentration in the LE through the remainder of this study. Effect of Electrolytes Chemistry. Stacking ratio and accumulation of sample ions in the ITP plug are highly affected by the concentration, i.e. conductivity, of the trailing and leading electrolytes.4,10,12 We performed an experimental parametric study focusing on the concentrations of TE and LE to empirically optimize the sample stacking ratio. Figure 3 shows the effect of TE (HEPES) concentration, CTE, on the ITP stacking ratio, C/Ci. We determine the maximum dye concentration in the plug, C, from the maximum of the Gaussian distribution fitted to the width-averaged intensity data. We varied CTE from 1.25 mM to 10 mM and kept the pH of the TE constant at 8.1 by adding Tris buffer at twice the concentration of HEPES. Here, the sample concentration in the TE reservoir, Ci, was 50 nM, and composition of the LE was fixed at 40 mM HCl, 80 mM Tris, and 3% PVP. We applied a constant current of 500 μA across a 45 mm long membrane and detected the fluorescent signal when the centerline of the plug reached 5 mm upstream of the LE reservoir. The data show that the stacking ratio decreases with increasing concentration of the TE by 4 fold over the range tested. Since the current in 5832

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry

Article

membrane resulting from evaporation should be avoided since it can reduce the stacking ratio. We also studied the dependence of the stacking ratio on the LE concentration by varying the HCl concentration, CLE. We kept the TE composition fixed at 2.5 mM HEPES and 5 mM Tris and the current density constant at 500 μA and found that the LE concentration does not have a significant effect on the ITP stacking ratio (data are not shown here). However, there are concentration limits that should be considered in designing the LE which are provided in the SI. Khurana and Santiago showed analytically and experimentally that in peak mode ITP, the stacking ratio is not a strong function of the LE concentration,12 which correlates well with our observations. In the goal of achieving LE and TE concentrations that result in the highest sample preconcentration along with the lowest heat generation-induced drying, which causes poor stacking ratios, we use a LE composition at 40 mM HCl and 80 mM Tris and TE composition of 2.5 mM HEPES and 5 mM Tris as standard solutions throughout the remainder of the experiments described in this work. Sample Zone Characteristics Change with Distance. In Figure 4, we demonstrate how the shape of the plug and stacking ratio change with location. Here, we are presenting results for a single representative ITP run on a 40 mm long membrane applied to a 500 μA constant current with our standard LE and TE solutions. Figure 4A shows five instantaneous images of the sample zone generation and migration from left to right toward the LE reservoir at (i) 5, (ii)

Figure 3. Plot of the stacking ratio, where the plug centerline is 5 mm upstream of the LE reservoir, as a function of TE (HEPES) concentration. pH of the TE maintained constant at 8.1 by Tris buffer. Solid line is a power function fitted to the experimental data, C/ Ci = 961.32CTE−0.62 (R2 = 0.96), showing that the stacking ratio is inversely proportional to the TE concentration. Here, the LE is 40 mM HCl, 80 mM Tris, and 3% PVP. Applied current is 500 μA, and length of the membrane is 45 mm. Each experimental data point represents three measurements, and the error bars denote a 95% confidence interval.

the system must be conserved, increasing the concentration of the TE reduces the local electric field in the TE zone and thus results in lower flux of sample ions into the ITP plug. We fit a power function to the data in Figure 3, C/C i = 961.32CTE−0.62 (R2 = 0.96). Analytical models describing physics of peak mode ITP suggest that the stacking ratio is inversely proportional to the TE concentration.12 We attribute this discrepancy between these models and our experimental observations to the effect of convective dispersion which was not accounted for in those models. At constant current density, a low concentration TE solution has lower conductivity, which results in higher electric field in the TE zone, ETE, due to Ohm’s law and the roughly linear dependence of the solution conductivity with the ionic concentration. Higher ETE increases the accumulation rate of the sample ions (per unit cross-section area), dNs/dt, in the plug, which leads to higher sample stacking. This rate equals the net electrophoretic flux of sample ions from the TE zone to the sample zone and can be expressed as12 dNS S S = (μTE − μTE )E TEC TE dt

(2)

μSTE

where is electrophoretic mobility of sample ions in the TE zone, and CSTE is the concentration of sample ions in the TE zone. Equation 2 correctly predicts that the stacking ratio can be increased, at constant current density, by increasing the ETE, i.e. lowering the CTE. However, we were not able to successfully run the ITP experiments at TE concentrations lower than 1.25 mM due to generation of high electric field in the TE zone, which burns the membrane and thus results in an open circuit and termination of ITP. Higher electric fields initiate excessive Joule heating, which, on a nitrocellulose membrane, can trigger sample evaporation since its surface is open to the ambient air. Excessive sample evaporation results in drying and ultimately burning of the membrane. As we will discuss later, drying of the

Figure 4. Change in focusing parameters of the plug as a function of its location normalized by the length of the membrane. (A) Snapshots taken from the sample zone at (i) 1, (ii) 30, (iii) 60, (iv) 90, and (v) 140 s after applying the electric field; (B) stacking ratio, sample concentration normalized by the initial sample concentration, and accumulated moles of sample in the plug normalized by the initial moles of sample in the TE reservoir (dashed line). Here, data are shown from a single ITP experiment using our standard electrolyte system. Applied current is 500 μA and length of the membrane is 40 mm. 5833

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry

Article

30, (iii) 60, (iv) 90, and (v) 140 s after applying the electric field. We normalized the location of the plug, x, by the total length of the membrane, Li, where x/Li = 0 is the TE reservoir and x/Li = 1 is the LE reservoir. The field of view in Figure 4A is x/Li = 0.25−1. The sample zone concentration increases as it travels through the membrane due to the electrophoretic influx of sample ions from the TE zone into the plug. Figure 4Ai shows the plug forming as a diffuse cloud of sample molecules. At this point, the plug is highly dispersed on the TE side (this is difficult to visualize with the current image settings) and exhibits a curvature in the direction of the ITP flow (toward right) which could be associated with the generation of a pressure gradient because of nonuniform axial EOF. At the middle of the membrane, Figure 4Aiii, the plug becomes upright with no curvature and then obtains a slight inverse curvature, toward the left, close to the LE reservoir as shown in Figure 4Av. Similar sample zone curvature trends have been observed in glass capillaries by Garcia-Schwarz et al.13 They associated this behavior to the internal pressure gradient generation in the capillary due to the axial mismatch of the EOF in the LE and TE zones. Note that the magnitude of the internal pressure generated in nitrocellulose membranes is not equivalent to capillaries because the membrane has a porous structure that is everywhere open to atmospheric pressure. However, nitrocellulose membranes are hydrophilic and thus will support some Laplace pressure. We calculate this pressure in our nitrocellulose membrane as 6 kPa (presented in the SI) which has the potential to support the EOF-induced internally generated pressure gradients. In Figure 4B, we plot the stacking ratio C/Ci and fractional number of moles N/Ni (number of moles of sample in the plug divided by the initial number of moles in the TE reservoir) as a function of the axial location of the ITP plug. The stacking ratio increases linearly with distance until it reaches 500 at x/Li = 0.6. The increase in stacking is a combination of a gradual increase in the number of sample moles and a sharp decrease in the width of the plug, as shown in Figure S-3. Further downstream, x/Li > 0.6, the stacking ratio roughly asymptotes. It has been shown theoretically and experimentally that the stacking rate remains constant through the entire peak mode ITP in a glass capillaries.12 We attribute this discrepancy to the convective sample dispersion caused by the strong presence of counter EOF which may have not been fully suppressed by PVP. Effect of Applied Current and Length of the Membrane. We showed that the stacking ratio and the amount of sample in the plug change with electrolyte concentration and the location of the ITP plug. Here, we investigate how electric field and length of the membrane influence these focusing parameters. We performed ITP experiments with the applied currents ranging from 100−850 μA on membranes with varying lengths of 35, 40, and 45 mm using our standard LE and TE solutions. For each experiment, we measured the stacking ratio and the fractional number of moles at a point 5 mm upstream from the LE reservoir. Figure 5A shows the stacking ratio as a function of applied current across the membrane. Each data point represents an average of at least four realizations, and the error bars denote a 95% confidence interval. For the 35-mm-long membrane, the stacking ratio increases with current and reaches the maximum value of 580 at 750 μA. By further increasing the current, the stacking ratio remains nearly constant, based on our Student’s t test analysis. Higher current density results in higher local electric field in each zone, which increases the counter EOF.

Figure 5. Plot of (A) stacking ratio, sample concentration normalized by the initial sample concentration, and (B) accumulated moles of sample in the plug normalized by the initial moles of sample in the TE reservoir, at a point where the centerline of the plug is 5 mm upstream of the LE reservoir, as a function of applied current for 35 mm (open circles), 40 mm (open diamonds), and 45 mm (open triangles) straight membranes and a 45-mm-long cross-shaped membrane (closed triangles). Here, we used our standard electrolyte system with the applied current of 500 μA. Error bars denote a 95% confidence interval. Solid and dashed lines are intended to enable the readers to follow the trend line of each data set.

Dispersion induced by EOF lowers the stacking ratio by reducing accumulation of sample ions and broadening the sample zone. We perform a detailed study of the width of the ITP plug as a function of the current, provided in the Supporting Information, that shows that for a 40 mm membrane, the width of the plug decreases linearly until 500 uA and then increases due to dispersion that is a result of Joule heating induced evaporation. As a result, the stacking ratio plateaus at higher current densities. Our data are in good agreement with Khurana and Santiago’s theoretical prediction that the sample stacking ratio increases linearly with current density in peak mode ITP.12 They also showed experimentally that the stacking ratio plateaus at higher currents densities when the effect of dispersion becomes significant. Note that, at a given condition, the time required to complete an ITP experiment reduces by increasing the applied current. For example, the time needed for an ITP experiment on 35 mm membrane at 100 μA is approximately 720 s, while it reduces to 110 s at 600 μA. 5834

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry

Article

physiochemical properties of the device, e.g., membrane type, target molecules, and time constraint of the experiments. Novel Membrane Designs. We showed that the sample dispersion due to the membrane drying reduces the ITP stacking ratio and fractional moles of the sample. When an electric current, I, flows through an electrolyte solution with conductivity σ in a channel with cross section A, Joule heating is produced, and for the electric power dissipated in a volume unit it scales as I2/Aσ. Joule heating is the main limitation in attempts to accelerate different electrophoretic analysis, including ITP, by using high currents or high voltages. Joule heating generates temperature gradients in the ITP zones, which results in some inhomogeneous physical and chemical properties, e.g. mobility, pH, density, etc.11 Further, it can cause sample evaporation on the open surface of the nitrocellulose membrane which at higher currents and a longer device length results in drying. Membrane drying disturbs electrophoretic migration of the ions and results in lower ITP preconcentration ratios. Therefore, excessive evaporation should be avoided in order to be able to apply higher currents across the membrane and achieve higher stacking ratio and sample extraction. We introduce a cross-shaped membrane design, as shown in Figure 1B, to mitigate the drying effects of evaporation and increase the stacking ratio of ITP. We hypothesize that by wetting the membrane through the two added wings we can maintain the hydration of the membrane so the sample molecules can migrate electrophoretically through the membrane. We placed the wings closer to the TE side because we observed enhanced drying in the TE zone due to its lower conductivity. In Figure 5A and B, we compare the stacking ratio and fractional number of moles in a 45-mm-long cross-shaped membrane to those of a straight membrane. The data show that we are able to apply higher currents and observe a significant increase in the stacking ratio and fraction of moles using the cross-shaped membrane. At 500 μA, the stacking increases by 17% compared to the straight 45 mm membrane and reaches 750 fold. We are able to apply currents up to 1 mA across the cross-shaped membrane and reach the maximum stacking ratio of 900 fold. Further increasing the current results in rapid and severe membrane drying and subsequent reduction in the stacking ratio. The fraction of moles accumulated follows similar trends as the stacking ratio showing that nearly 60% of initial sample can be extracted at 1 mA. We showed here that by using cross-shaped membrane structures we are able to run ITP at higher currents, which improves the stacking ratio, and sample extraction. Moreover, ITP can be run at much shorter times, less than 90 s, by operating at these high currents. Battery Powered ITP. One of the main drivers behind the development of paper based microfluidic devices is their relatively low cost and simplicity compared to microfabricated channel devices. In the interest of an instrument free device, we have performed ITP experiments using commercially available button style and AA batteries and miniature high voltage DC supply in the place of research grade high voltage power supplies. The use of portable and inexpensive power supplies may provide an opportunity to integrate ITP into point-of-care diagnostics. For a typical ITP experiment on a straight 40-mm-long membrane, we applied 500 μA of current and sourced average of 440 V from a power supply for 4 min. This ITP experiment requires 220 mW of power and consumes 52 J (or 9 mAh at 1.55 V) of energy. A typical smart phone battery stores 4300 mAh of energy, an alkaline AA battery stores 2122 mAh, and

When we increased the length of the membrane to 40 mm, we observed an increase in the maximum stacking ratio to 760 at 500 μA. In a longer membrane, more sample ions accumulate in the plug because they have more time to reach the sample zone, resulting in a higher stacking ratio. However, in the 40 mm membrane, the stacking ratio decreases at currents higher than 600 μA because of the dominant effect of dispersion caused by sample evaporation. Joule heating, which is directly proportional to the applied current, triggers evaporation of the solutions due to the open surface of nitrocellulose and at higher currents leads to a dry membrane. Drying, which is more prominent in the low conductivity TE zone, has the potential to disturb influx of the sample ions into the ITP plug, resulting in a lower stacking ratio. Moreover, dry regions of the membrane have higher electrical resistance, thus local electric field increases in order for the current to be conserved, resulting in higher sample dispersion. In order to further investigate the effect of length on the stacking ratio, we increased the length to 45 mm; however, we do not see an increase in stacking ratio compared to the 40 mm membrane. This can be explained by the high electrical resistance of the 45-mm-long membrane, which increases electric field across the membrane and thus in each ITP zone. Higher electric field in ITP zones increases dispersion due to EOF and results in a lower stacking ratio. Similar to what we observed for the 40 mm membrane, currents in excess of 500 μA reduce the stacking ratio as a result of dispersion due to evaporation. Note that for 45 mm, membrane evaporation starts at lower currents, ∼500 μA, compared to the 40-mm-long membrane because of its higher resistance. In Figure 5B, we show the fractional number of moles as a function of applied current for the same conditions as in Figure 5A. The fractional number of moles is an indication of the amount of sample extracted from the TE reservoir and focused in the ITP plug. The fractional number of moles follows roughly the same trend as the stacking ratio. For each length tested, we see a region where the fractional number of moles increases linearly with current and then reduces at higher currents due to the effect of different sources of dispersion, i.e. nonuniform EOF and evaporation. Analytical and experimental studies on peak mode ITP suggests that the fractional number of moles of the sample does not depend on the applied current,12 but these models do not account for the convective dispersion due to counter EOF which cannot be fully suppressed in nitrocellulose membrane. These models were conducted for glass capillaries, which are exempt from sample evaporation due to heating. Figure 5B shows that increasing the length of the membrane from 35 mm to 40 mm improves the fractional number of moles by 10% ,while further increasing the length to 45 mm results in an inverse trend due to the dominant effect of dispersion. We were able to achieve a 760 fold stacking ratio and sample extraction of 50% by applying a 500 μA current on a 40-mm-long membrane. These data suggest that there is an optimum in both applied current and device length in respect to the maximum stacking ratio. Higher applied current leads to higher stacking ratio, more sample extraction, and faster preconcentration. Increasing the membrane length is also desirable since it improves the stacking ratio and fractional number of moles. However, there is a limit for increasing these parameters to avoid negative impacts of sample dispersion due to evaporation and EOF. In order to obtain the highest sample preconcentration, one should find optimum length and current based on the 5835

dx.doi.org/10.1021/ac500780w | Anal. Chem. 2014, 86, 5829−5837

Analytical Chemistry

■

SUMMARY AND CONCLUDING REMARKS We have demonstrated preconcentration of Alexa Fluor 488 on a nitrocellulose membrane using peak mode ITP. Up to a 900 fold increase in the initial sample concentration and 60% extraction from 100 μL of the sample were achieved by introducing a novel cross-shaped membrane that reduces sample evaporation at high currents. Using a relatively large volume of sample compared to the sample volumes used in microfluidic devices suggests that ITP on paper may be useful in applications where the analyte of interest is dilute and the sample can be large. We performed detailed studies on how variations in different parameters including chemistry of the electrolytes, applied current, and length of the device affect the ITP stacking ratio. For example, low TE concentration, high current density, and long membrane length result in higher stacking ratio and sample extraction. In an effort to reach higher sample extractions, we introduced 5 μL of the sample on the middle of the membrane between the TE and LE, which can easily be done using open surface paper based device. Our measurements (not shown here) show that 80% of sample molecules can be extracted and focused using this technique. Moreover we showed that ITP on a nitrocellulose membrane can be powered and run several times by a small button battery with comparable performance, which suggests that ITP could be integrated to a portable point-of-care diagnostic tests. Integrating ITP into lateral flow immunoassays for faster and more sensitive detection of biomolecules is an ongoing task in our research group.

typical silver oxide button battery stores 170−200 mAh, which are all several time more than the power required for the ITP experiment. We designed a battery powered miniature high voltage DC supply. Table 1 summarizes the stacking ratios obtained on a Table 1. Stacking Ratios Achieved Using Batteries and Conventional Power Supply to Power ITPa power source

applied voltage (V)

conventional power supply AA battery conventional power supply button battery

350 350 250 250

C/Ci 638.60 564.07 607.00 541.13

± ± ± ±

Article

20.63 48.58 20.18 31.60

a

Each data point represents three measurements with the uncertainties calculated with a 95% confidence interval.

40-mm-long membrane using a miniature power supply powered by an AA battery and a button battery as well as with a conventional power supply. The battery powered supply outputs a variable voltage that increases with time because the resistance of the ITP assay is changing with time as the low conductivity trailing electrolyte replaces the leading electrolyte (see Supporting Information Figure S-5). The AA and button batteries operate at 350 and 250 V average voltages, respectively. When the AA battery 1.55 V input voltage is applied, we would expect an output voltage of 600 at the open circuit; however, since the voltage converter is limited to 0.1 W (under the 1.4 MΩ rated load) we achieve a voltage of 150− 450 V that varies in time with the resistance of the ITP assay. We observe a similar behavior with the button battery, but its power capacity is lower than the AA because its performance is further limited by the batteries’ inherent voltage−current output behavior that causes a more significant drop in its output voltage (