Expanding the Capabilities of Microfluidic Gradient Elution Moving

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Expanding the Capabilities of Microfluidic Gradient Elution Moving Boundary Electrophoresis for Complex Samples Elizabeth A. Strychalski,*,† Alyssa C. Henry,‡ and David Ross† † ‡

Biochemical Science Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States Applied Research Associates, Incorporated, Arlington, Virginia 22203, United States

bS Supporting Information ABSTRACT: Gradient elution moving boundary electrophoresis (GEMBE) is a robust, continuous injection separation technique that uses electrophoresis to drive electrically charged analytes into a capillary or microfluidic channel for detection, while opposing electroosmosis and controlled variable pressuredriven flow prevent other sample components—for example, cells, proteins, or particulates in complex samples that can interfere with analysis—from entering the channel. This work expands the sample-in/answer-out analytical capabilities of GEMBE for complex samples by demonstrating the quantitative analysis of anions, implementing aqueous background electrolyte (BGE) solutions at neutral pH, and introducing the use of additives to the sample solution to optimize performance. Dirt was analyzed quantitatively, with the sole preparatory step of suspension in an aqueous BGE solution at neutral pH, for dissolved chloride, nitrite, nitrate, sulfate, and oxalate using GEMBE with capacitively-coupled contactless conductivity detection. In addition to altering the pH of the BGE solution, optimization of the analysis of dirt and whole blood was achieved using various commercially available additives. These results, taken together with previous demonstrations of GEMBE for the analysis of complex samples, underscore the uncomplicated versatility of GEMBE, facilitate effective analysis of biological complex samples using BGE solutions at physiological pH, and offer a sufficient set of techniques and tools to build a foundation for the analysis of a broad range of complex samples.

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nalytical techniques capable of characterizing complex samples are needed for a variety of applications, such as routine clinical screening of biological samples for healthcare applications, ensuring the security of food and other consumer products, and testing environmental samples for pollutants.1 Ideally, means for these analyses would be inexpensive, disposable, and portable, which are advantages associated with miniaturized fluidic technologies that form the basis for lab on a chip analytical platforms.2 Moreover, these complicated analytical tasks involve interrogation of complex samples consisting of mixtures containing many disparate components, for example, cells, proteins, or particulates, that may hinder or preclude chemical analysis of the sample. Although miniaturized analytical methods tend to be adversely sensitive to these sample components, which often clog or foul microfluidic structures, a new miniaturized fluidic analytical method, gradient elution moving boundary electrophoresis (GEMBE),3,4 is well-suited to rapid, reproducible, quantitative, and inexpensive analysis of complex samples.5 GEMBE is a continuous injection separation technique performed using a simple miniaturized fluidic geometry consisting of two fluid reservoirs—one for the sample to be interrogated and another containing background electrolyte (BGE) solution—connected by a microscale fluidic conduit, such as a microfabricated channel or capillary (Figure 1a c). Electrophoretic transport initiated via electrodes inserted into each of the two reservoirs r 2011 American Chemical Society

carries the electrically charged analytes from the sample reservoir and into the microfluidic channel for detection. A controlled, variable, pressure-driven counterflow provides selectivity for the analytes. During a GEMBE separation, the applied pressure is reduced gradually, and an analyte enters the microfluidic channel for detection when its electrophoretic velocity overcomes the opposing electroosmotic and pressure-driven flows. This process is allowed to progress until each analyte of interest has been detected, usually via fluorescence,3,6 channel current,7 or capacitively coupled contactless conductivity detection (C4D)5,8 10 (as in the work described here). The salient feature of GEMBE most relevant to the investigation of complex samples is the pressure-driven transport, which prevents sample components other than the analytes of interest from entering the microfluidic channel and compromising its performance.11 This simple mechanism for addressing the challenges of analyzing complex samples using microfluidic technology avoids both laborious and methodical sample preparation prior to introduction on-chip and the complexity of serially integrated microfluidic structures, such as filters or extraction elements. The pressure and its gradient in time can also be altered easily, without the need to construct new device Received: May 9, 2011 Accepted: June 17, 2011 Published: July 18, 2011 6316

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Figure 1. GEMBE analysis of complex samples with C4D and minimal sample preparation. (a c) Schematic of the experimental apparatus (not to scale) shows an electrically grounded sample reservoir at ambient pressure and a reservoir for background electrolyte (BGE) solution held at high voltage and a controlled variable pressure, connected by a microfluidic capillary with a C4D detector. Arrows illustrate the directions and relative magnitudes of electrophoretic transport and opposing electroosmotic and variable pressure-driven transport. This bulk counterflow provides selectivity for the analytes and prevents particulates and other matrix interfering agents (black dots) from entering the capillary. (b) An electrically charged analyte (yellow) enters the microfluidic channel for detection as a stepwise increase in conductivity, when its electrophoretic mobility overcomes the counterflow. (c) The pressure is further reduced until all analytes (yellow and orange) have entered the capillary for detection. Photographs show dirt (scale bar: 1.7 cm) (d) and whole blood (scale bar: 0.7 cm) (e) sample solutions.

geometries, allowing rapid optimization of separation parameters in real time.11 GEMBE is readily applicable to the analysis of real-world, complex samples, when the analytes of interest and problematic matrix interfering agents exhibit opposing electrical charges. Separation parameters can then be chosen so that electrophoretic, electroosmotic, and pressure-driven transport all contribute to drive the interfering agents away from the microchannel entrance, without restricting the movement of analytes for detection. Such was the case for the first demonstration of GEMBE analysis of complex samples by the authors.5 This previous study verified the utility of GEMBE for quantitative measurements of various cations in complex samples, including dirt, milk, estuarine sediment, coal fly ash, leaves, and serum, that contained anionic proteins, particulates, and other matrix interfering agents. The current work addresses the more difficult task of establishing GEMBE as a viable technique to analyze complex samples containing analytes and contaminants with like electrical charges. In particular, the results presented here demonstrate methods for the GEMBE analysis of anions in complex samples, including dirt and whole blood, containing anionic matrix interfering agents. For the case in which the electrophoretic mobilities of the anionic analytes were significantly faster than that of the anionic particulates in a sample, GEMBE analysis of dirt is performed here similarly to the methods described previously.5 Specifically, dirt is suspended in an aqueous buffer at neutral pH and analyzed quantitatively for dissolved chloride, nitrite, nitrate, sulfate, and oxalate. The physiological conditions of the BGE solution used here further extend the applicability of GEMBE to the interrogation of broad classes of complex biological samples. To analyze anions with electrophoretic mobilities similar to or slower than that of interfering agents in a sample, GEMBE requires modification for reliable results. For the analyses presented here, such analytes include phosphate, lactate, adenosine triphosphate (ATP), or adenosine diphosphate (ADP) in dirt and whole blood. The simplest modification involves selection of a BGE solution with a lower pH, such that the electrophoretic

mobilities of particulates and other matrix interfering agents are altered to prevent entrance into the microchannel. This approach, however, may not be satisfactory for many assays in which a BGE solution at a particular pH is required, for example, a near-neutral pH for many biologically relevant assays. To address this need, the addition of various chemical additives to the sample solution is introduced here. The suitability of several common, commercially available dynamic surface coatings is examined for use with GEMBE analysis of complex samples. These additives are hypothesized to coat particulates in the sample, thereby modifying their electrophoretic mobilities. In this way, the elution of particulates is effectively slowed relative to the analytes of interest. The additives contact the sample reservoir only, without entering the microchannel, preserving the ability for GEMBE to rapidly analyze different samples simply by exchanging the fluid in the sample reservoir. This tactic increases the flexibility of GEMBE with minimal added complication and allows the selection of a BGE solution best suited to the analysis at hand.

’ MATERIALS AND METHODS Experimental Apparatus. A schematic of the experimental apparatus is shown in Figure 1a c. A microchannel consisting of a 5-cm-long fused-silica capillary with nominal outer and inner diameters of 360 and 15 μm, respectively, spanned two reservoirs for fluid. The smaller reservoir was machined from poly(oxymethylene), held 200 μL of sample dissolved or suspended in BGE solution for analysis, and was kept at ambient pressure. The larger, sealed reservoir was machined from either poly(sulfone) or poly(etherimide), held 2 mL of BGE solution, and was regulated at a variable pressure using a pressure controller (series 600, Mensor) backed by pressurized helium. The capillary was inserted ∼1 mm into the sample reservoir through a tight-fitting, drilled hole, then through a contactless conductivity detector (TraceDec, Innovative Sensor Technologies), and finally, ∼4 mm into the BGE reservoir through a miniature compression 6317

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Figure 2. Experimental data demonstrating quantitative GEMBE analysis of dissolved anion content in BGE solution and dirt sample solution. (a) Representative raw data showing stepwise increases in detected conductivity due to the elution of chloride (1), nitrite (2), nitrate (3), sulfate (4), oxalate (5), sulfite (6), and phosphate (8) of known concentration added to BGE solution (a) and dirt (c), which was prepared at 54.7 mg/mL in BGE solution. Fifty-one point Savitsky-Golay time derivatives of (a) and (c) display the BGE solution (b) and dirt sample solution (d) data as peaks. The presence of bicarbonate (7) is due to dissolution of carbon dioxide from the ambient atmosphere. The irregular peak shape of sulfate (6) and evidence for particulate entrance into the microchannel (*) suggest that an alternate BGE solution, for example, with a lower pH, or additives to the sample solution to prevent particulates from interfering with analysis would be beneficial. These analyses used a start pressure of 10 kPa and a pressure gradient of 50 Pa/s. Data have been shifted vertically for clarity.

fitting (LabSmith) machined into the reservoir for BGE solution. The detection point was ∼20 mm from the capillary entrance in the sample reservoir. Platinum electrodes in the reservoirs supplied +2000 V dc (PS350, Stanford Research Systems). The detector settings were frequency, 2 high; voltage, 0 dB; gain, 200%; offset, 19; filter, slow; and data acquisition rate, 19.8 Hz. The fluidic and detector portions of the apparatus were encased in poly(methyl methacrylate) for mechanical and thermal stability. The apparatus was controlled and the data recorded using custom (LabView, National Instruments) and vendorsupplied (TraceDec Monitor 0.07a) software. Materials for custom machined parts were obtained from McMaster-Carr. Note that the results presented here were obtained over approximately 1 year using different capillaries with identical nominal properties. As a result, the optimal starting pressure varied between sets of analyses, which may be attributed to slight differences in the electroosmotic properties and inner diameter of the various capillaries. Sample Preparation. BGE solution of 100 mmol/L bis-Tris (14879, Fluka) and 100 mmol/L HEPES (54457, Fluka) at pH 7.1 was prepared using 18.2 MΩ cm water and filtered using a 0.2

μm poly(propylene) filter. Dirt was obtained from beneath several oak trees on the Gaithersburg campus of the National Institute of Standards and Technology and was similar to that analyzed by the authors for dissolved cation content using GEMBE.5 CD-1 mouse whole blood with Na heparin was purchased from Innovative Research. After suspension or dilution in BGE solution, dirt and whole blood sample solutions (Figure 1d,e) were vortexed briefly and pipetted directly into the sample reservoir for analysis without further preparation or sample cleanup. Stock solutions of sodium chloride (Mallinckrodt), sodium nitrite (Mallinckrodt), potassium nitrate (Allied Chemical), sodium sulfate (Mallinckrodt), sodium oxalate (JT Baker Chemical), sodium sulfite (Mallinckrodt), and potassium phosphate (Sigma) were prepared separately in BGE solution at 100 mmol/L, while lactate (Sigma), ATP (Sigma) and ADP (Sigma) were prepared separately at 10 mmol/L in BGE solution. These stock solutions were added as necessary to the dirt and whole blood sample solutions to achieve the desired final concentration of each of the associated anions for a given analysis. 6318

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Figure 3. GEMBE analysis of anionic analytes in dirt sample solutions using additives to suppress the elution of particulates. Representative data showing conductivity steps and corresponding peaks are given for dirt sample solutions without (black) and with (gray) EOTrol HR (a), EOTrol LN (b), PVP (c), and DDAB (d) added to the sample solution. These data focus on the effects of each additive on data quality for analytes with mobilities similar to or slower than particulates (*) in the sample solution. Identified analytes are bicarbonate (1), phosphate (2), ATP (3), ADP (4), and an unknown constituent of PVP (5). Data in (a) were obtained using an 83 mg/mL dirt sample solution, 5% (v/v) EOTrol HR, a starting pressure of 10 kPa, and a pressure gradient of 100 Pa/s. Data in (b) used an 89.6 mg/mL dirt sample solution with 50 μmol/L phosphate, ATP, and ADP; 2.4% (v/v) EOTrol LN; a starting pressure of 2.5 kPa; and a pressure gradient of 100 Pa/s. Analyses in (c) used an 89.2 mg/mL dirt sample solution with 50 μmol/L potassium phosphate, ATP, and ADP; 0.38% PVP (w/v); a starting pressure of 3.5 kPa; and a pressure gradient of 100 Pa/s. Data in (d) used an 82 mg/mL dirt sample solution and an 83 mg/mL dirt sample solution with 100 μmol/L DDAB, a starting pressure of 10 kPa, and a pressure gradient of 100 Pa/s. Care should be taken that contributions to the data from the additive itself, for example, analyte (5), not obscure analytes of interest. The addition of EOTrol LN most effectively diminished the effects of particulates on the data. These results establish the compatibility of GEMBE and additives, which can be used to optimize the analysis of complex samples and allow for the simultaneous measurement of analytes with an expanded range of mobilities for a given BGE solution. Data have been shifted vertically for clarity.

For analyses in which additives were employed, stock solutions of EOTrol high reverse (EOTrol HR, Target Discovery) and EOTrol low normal (EOTrol LN, Target Discovery) were added directly to the dirt and whole blood sample solutions in the sample reservoir. A stock solution of 25% (w/v) poly(vinylpyrrolidone) (PVP, 40 kDA, Fluka) was prepared in 18.2 MΩ cm water. Didecyldimethylammonium bromide (DDAB, Sigma) was dissolved in BGE solution at 1 mmol/L. These stock solutions of PVP and DDAB were also added directly to the sample solution for dirt and whole blood analyses using these additives. Experimental Procedure. A typical experimental analysis began with the BGE solution reservoir held at 30 kPa while the sample reservoir was at ambient pressure. The sample reservoir was rinsed with BGE solution and filled with 200 μL of dirt or whole blood sample solution for analysis. The voltage was applied, and the pressure was simultaneously reduced slightly to the starting pressure for that analysis and held constant for ∼13 s. The pressure was subsequently decreased further at a specified rate (typically, 50 Pa/s or 100 Pa/s) until all analytes of interest were detected, at which point the pressure was increased to 30 kPa for ∼10 s, and the voltage was turned off.

The apparatus was held in this configuration for least 1 min to flush the microchannel with BGE solution prior to the next analysis. Data were indistinguishable for results obtained by simply pipetting the sample solution into the sample reservoir, mixing by pipetting the solution in and out of the sample reservoir vigorously, or mixing during analysis with a magnetic stir bar operating at the bottom of the sample reservoir. The resulting data was analyzed according to the procedure outlined in the Supporting Information. For each set of experimental conditions, separate experiments were performed to identify analytes associated with particular detected stepwise changes in conductivity. The concentration of each anion in the sample solution was increased individually, and the correlated detector response was identified (data not shown).

’ RESULTS AND DISCUSSION Dirt was chosen as a representative complex sample whose quantitative analysis embodies many of the general challenges associated with the miniaturized analysis of anions in complex samples. Figure 2 presents representative raw detector signals and associated time derivatives. The former shows analytes as a 6319

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Table 1. Additive Performancea additive sample dirt whole blood

EOTrol HR b d

++

EOTrol LN d

++

b

PVP

DDAB

c

b

+

b

c

+

a

Additives to the dirt and whole blood sample solutions were tested for their efficacy in reducing noise in the data attributable to particulates and other matrix interfering agents entering the microchannel during GEMBE analysis. b , no improvement compared to analysis without additive. c +, some improvement over analysis without additive. d ++, significant improvement over analysis without additive, with data quality comparable to analysis of samples without particulates or interfering agents.

series of stepwise increases in conductivity, whereas the latter yields peaks for facile comparison with more conventional separation methods and analyses. Calibration and standard curves were constructed for anionic analytes at various concentrations added to BGE solution and dirt suspended in BGE solution, respectively (Figure 2, Supporting Information Figure S-1). Linear fits to the calibration and addition data were used to calculate the concentration and apparent recoveries of chloride, nitrite, nitrate, sulfate, and oxalate in the dirt sample solutions (Supporting Information Figure S-1 and Table S-1).12 This demonstrates quantitative GEMBE analysis of a complex sample in which analytes and contaminants exhibit like electrical charges. These results were obtained for anionic analytes that eluted faster than the anionic particulates. Consequently, these faster analytes appeared clearly in the detector response with good signal-to-noise ratios, enabling reliable quantitative analysis using GEMBE without modification of the basic experimental procedure or apparatus from the previous demonstration examining cations in complex samples.5 Here, the decreasing pressure gradient can simply be halted after all analytes of interest have been detected and before the slower particulates enter the microfluidic channel from the sample reservoir. For analytes with similar or slower mobilities, however, such as those eluting near the end of each analysis in Figure 2, a diminished signal-tonoise-ratio due to the coelution of particulates hampered reliable quantification. For dirt analyses, this increased baseline noise was highly irreproducible, observed exclusively at times later than the detection of phosphate, and when present, manifested most often as discrete downward “spikes” in the detector signal (Figures 2 4). The detrimental effects of the spikes were eliminated by choosing a BGE solution with a reduced pH so that particulates were no longer driven electrophoretically into the microchannel during analysis (Supporting Information Figure S-2). However, it is often necessary to work at a particular pH, for example, the near neutral pH required for enzyme assays,7 analyses of complex biological mixtures that benefit from the prevention of cell rupture in the sample, or to provide selectivity for analytes whose acid dissociation constants call for analysis at neutral or higher pH. The use here of a BGE solution with neutral pH supports the utility of GEMBE for interrogating samples of biological relevance, as many of these matrices contain significant quantities of cells, proteins, particulates, and other substances that often complicate analysis or may be adversely affected by typical sample preparation. Whole blood was chosen as a quintessential biological complex sample to demonstrate the ability for GEMBE

Figure 4. GEMBE analysis of anionic analytes in whole blood using additives to suppress the elution of matrix interfering agents. Representative data for whole blood diluted 100 in BGE solution are given for whole blood without (black) (a c) and with the addition of EOTrol HR (gray) (a), EOTrol LN (gray) and PVP (light gray) (b), and DDAB (gray) (c). These results focus on the effects of each additive on data quality for analytes with mobilities similar to or slower than cells, particulates, or other matrix interfering agents (*) in the sample solution. Identified analytes are bicarbonate (1), phosphate (2), lactate (3), and unknown components of PVP (4) and EOTrol LN (5). Analyses in (a) used 5% (v/v) EOTrol HR, a starting pressure of 10 kPa, and a pressure gradient of 100 Pa/s. Data in (b) were obtained using 5% (v/v) EOTrol LN, 1% (w/v) PVP, a starting pressure of 2 kPa, and a pressure gradient of 100 Pa/s. Data in (c) used 100 μmol/L DDAB, a starting pressure of 10 kPa, and a pressure gradient of 100 Pa/s. Degradation of the data due to the detection of sample contaminants (*) was mitigated most effectively with EOTrol HR. This technique could be applied generally to greatly expand the range of complex samples addressable by GEMBE. Data have been shifted vertically for clarity. 6320

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Analytical Chemistry to successfully detect anionic species with no sample preparation beyond dilution in BGE solution. However, blood analyses also suffered from spikes in the data that occurred at ∼1 spike/s, often overlapped, and obscured analyte signals at elution times longer than that of phosphate. Similarly to the dirt analyses, spikes associated with particulates or other matrix interfering agents were not observed to elute before phosphate. Because the use of an alternative BGE solution can be prohibitive for comprehensive analyses of whole blood and other complex biological samples, a different strategy was pursued to improve analytical results. A small amount of a dynamic surface coating was added to the sample solution prior to analysis. Such additives are hypothesized to facilitate GEMBE analysis of complex samples by coating particulates to slow or reverse their electrophoretic mobility, thereby preventing these materials from entering the microchannel during analysis. The current work examined four additives commonly used to coat fused silica capillaries to modify electroosmotic flow during capillary zone electrophoresis separations: EOTrol HR, EOTrol LN, PVP, and DDAB. Representative GEMBE analyses of dirt and whole blood sample solutions with these various additives are shown in Figures 3 and 4, respectively, with attention focused on data near the elution of phosphate and later. For some experiments, lactate, ATP, and ADP were included in the sample solutions as additional analytes with elution times longer than that of phosphate. The performance of each additive was determined by comparison with results obtained for the same sample without additive, analyzed on the same day, and using the identical microchannel and experimental apparatus (Table 1). Overall, the proprietary EOTrol additives performed better than the more commonly used, nonproprietary additives. Neutral additives more effectively improved GEMBE analysis of dirt, whereas cationic additives gave better results with whole blood analyses. Due to the sporadic appearance of particulates in data for dirt analyses, further experimental results are shown for analyses of dirt sample solutions without additives (Supporting Information Figures S-4, S-5, and S-6) and containing EOTrol HR (Figure S-3), EOTrol LN (Figure S-4), PVP (S-5), and DDAB (S-6). These results mark a new application for dynamic surface coatings, which are added to the sample solution in the sample reservoir only, instead of coating a capillary surface to modify electroosmotic flow. Many additives beyond those considered here have been employed for surface modification of capillaries and microchannels,13 15 and any of these could be compatible with and easily utilized to facilitate and optimize GEMBE analysis of a complex sample of interest. Considering the multitude of dynamic surface coatings available with a wide variety of properties and the robustness of GEMBE itself, the use of additives is expected to greatly expand the range of complex samples addressable by GEMBE.

’ CONCLUSIONS GEMBE has proven to be an extremely robust analytical method, delivering quantitative results with minimal sample preparation across a broad range of BGE solution conditions and complex sample matrices. The ability of GEMBE to interrogate complex samples in a sample-in answer-out fashion, in particular, distinguishes it from many other miniaturized techniques. The analytical relevance of GEMBE is expanded by the ability to measure both the dissolved cationic and, as

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demonstrated in this work, anionic content of complex samples. The use of additives enables the simultaneous analysis of analytes with a greater range of mobilities for a given complex sample and BGE solution and is expected to be applicable to the optimization of GEMBE analyses of cationic analytes, in addition to anionic analytes, in complex samples. These additives also confer the freedom to choose a BGE solution best suited to a given complex sample, for example, physiological buffers at neutral pH for biological samples. In summary, the use of additives introduced here is expected to complement techniques employed in previous GEMBE analyses and form the foundation for the reliable, quantitative GEMBE analysis of an expanded range of candidate complex samples and analytes. Regarding the GEMBE apparatus itself, C4D increases the portability and ease of use of the analytical apparatus9 as well as reducing its overall cost relative to other common detection schemes. For these reasons, GEMBE could play an important role in meeting analytical needs for realworld samples in time-critical applications, such as screening for security threats, and in resource-limited situations, such as humanitarian relief efforts and natural disaster zones.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Address: Biochemical Science Division, National Institute of Standards and Technology, 100 Bureau Drive, Mail Stop 8313, Gaithersburg, MD 20899, USA. Telephone: 001.301.975.5951. Fax: 001.301.330.3447. E-mail: [email protected].

’ ACKNOWLEDGMENT This work was performed while author E.A.S. held a National Research Council Research Associateship Award. Certain commercial equipment, instruments, or materials are identified to adequately specify the experimental procedure. Such identification implies neither recommendation or endorsement by the National Institute of Standards and Technology nor that the materials or equipment identified are necessarily the best available for the purpose. ’ REFERENCES (1) Crevillen, A. G.; et al. Talanta 2007, 74 (3), 342–357. (2) Mark, D.; et al. Chem. Soc. Rev. 2010, 39 (3), 1153–1182. (3) Shackman, J. G.; Munson, M. S.; Ross, D. Anal. Chem. 2007, 79 (2), 565–571. (4) Vyas, C. A.; Flanigan, P. M.; Shackman, J. G. Bioanalysis 2010, 2 (4), 815–827. (5) Strychalski, E. A.; Henry, A. C.; Ross, D. Anal. Chem. 2009, 81 (24), 10201–10207. (6) Shackman, J. G.; Ross, D. Anal. Chem. 2007, 79 (17), 6641–6649. (7) Ross, D.; Kralj, J. G. Anal. Chem. 2008, 80 (24), 9467–9474. (8) Guijt, R. M.; et al. Electrophoresis 2004, 25 (23 24), 4032–4057. (9) Kuban, P.; Hauser, P. C. Electrophoresis 2009, 30 (1), 176–188. (10) Flanigan, P.; Ross, D.; Shackman, J. G. Electrophoresis 2010, 31 (20), 3466–3474. (11) Munson, M. S.; Meacham, J. M.; Locascio, L. E.; Ross, D. Anal. Chem. 2008, 80 (1), 172–178. 6321

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