Toward a Boron-Doped Ultrananocrystalline Diamond Electrode

Article pubs.acs.org/ac

Toward a Boron-Doped Ultrananocrystalline Diamond ElectrodeBased Dielectrophoretic Preconcentrator Wenli Zhang and Adarsh D. Radadia* Institute for Micromanufacturing, Center for Biomedical Engineering and Rehabilitation Sciences, Chemical Engineering, Louisiana Tech University, Ruston, Louisiana 71272, United States S Supporting Information *

ABSTRACT: This paper presents results on immunobeadsbased isolation of rare bacteria and their capture at a borondoped ultrananocrystalline diamond (BD-UNCD) electrode in a microfluidic dielectrophoretic preconcentrator. We systematically vary the bead surface chemistry and the BD-UNCD surface chemistry and apply dielectrophoresis to improve the specific and the nonspecific capture of bacteria or beads. Immunobeads were synthesized by conjugating antibodies to epoxy-/sulfate, aldehyde-/sulfate, or carboxylate-modified beads with or without poly(ethylene glycol) (PEG) coimmobilization. The carboxylate-modified beads with PEG provided the highest capture efficiency (∼65%) and selectivity (∼95%) in isolating live Escherichia coli O157:H7 from cultures containing 1000 E. coli O157:H7 colony-forming units (cfu)/mL, or ∼500 E. coli O157:H7 and ∼500 E. coli K12 cfu/mL. Higher specificity was achieved with the addition of PEG to the antibody-functionalized bead surface, highest with epoxy-/sulfate beads (85−86%), followed by carboxylate-modified beads (76−78%) and aldehyde-/sulfate beads (74−76%). The bare BD-UNCD electrodes of the preconcentrator successfully withstood 240 kV/m for 100 min that was required for the microfluidic dielectrophoresis of 1 mL of sample. As expected, the application of dielectrophoresis increased the specific and the nonspecific capture of immunobeads at the BD-UNCD electrodes; however, the capture specificity remained unaltered. The addition of PEG to the antibody-functionalized BD-UNCD surface had little effect on the specificity in immunobeads capture. These results warrant the fabrication of electrical biosensors with BD-UNCD so that dielectrophoretic preconcentration can be performed directly at the biosensing electrodes.

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Microfluidic DEP has been successfully used with polystyrene beads,18−20 bacteria,18,21−23 yeast,24 virus,25 and DNA.26,27 A variety of electrode designs have been used to implement DEP on a chip. Interdigitated electrodes,18−27 and its variations such as castellated,28,29 sawtooth,30 off-chip electrode,18,31 and extruded,32−34 are planar in design and easy to assemble with microfluidics. However, the magnitude of DEP force decreases exponentially with distance from the electrode plane;35 hence, such devices use shallow channels (≤15 μm) and a low flow rate (≤1 μL/min).36 These limitations can be overcome using a set of parallel electrodes as the top and the bottom of the microfluidic channel.37−39 Regardless the configuration, the electrodes are typically made of gold, platinum, or indium−tin oxide and are insulated with silicon dioxide or silicon nitride to prevent damage to the electrodes resulting from Joule heating or electrolysis. Thus, DEP has not been applied directly for durations greater than 15 min at bare electrodes that are used in cyclic voltammetry,

iniaturized biosensors hold great promise for rapid, sensitive, and label-free field detection of pathogens for food safety,1,2 medical diagnosis,3 and epidemiology.4 Pushing the biosensor design to extremely small sizes suggests increased sensitivity in pathogen detection; however, this implies that the associated fluidics also have to be scaled down in order to reduce the diffusive and the convective transport times of the pathogens to the sensor, and therefore the sample volumes have to be decreased or the analysis time has to be increased. Alternatively, off-chip sample preparation can be used to isolate pathogens from the sample and preconcentration schemes can be integrated on-chip to increase the pathogen capture at sensor. On-chip preconcentration have previously been reported using dielectrophoresis (DEP),5−8 magnetophoresis,9−12 acoustophoresis,13,14 optophoresis,15 and hydrophoresis.16,17 DEP, being an electrical technique, is more attractive for portable applications and field deployment. In DEP, a time varying, nonuniform electric field induces polarization at the cell surface and results in the translational motion of the cells suspended in a fluid. Combining DEP with microfluidics is practical because only a few volts are necessary to generate the sufficient field strengths for moving cells or bacteria on a chip. © XXXX American Chemical Society

Received: August 22, 2015 Accepted: February 2, 2016

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DOI: 10.1021/acs.analchem.5b03227 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

magnetic beads. The polystyrene beads were separated from bacteria by centrifuging at 2000 rpm for 9 min and washed twice with PBS. The magnetic beads were separated using a magnet block. Negative control samples were created by eliminating the bead addition step; this allowed enumerating the bacteria that settled during the selective centrifuging of beads and beads with bacteria or their magnetic separation. The beads were then plated on LB or MacConkey sorbitol (SMAC) agar plates for 12 h at 37 °C to determine the efficiency and selectivity attained in pathogen isolation. The LB agar plates are nonselective and thus allowed enumeration in capture experiments with isolate cultures. The SMAC agar allows differentiation between E. coli O157:H7 and E. coli K12 captured from the cocultures. E. coli O157:H7 produces clear colonies on the SMAC agar while E. coli K12 produces pink colonies. BD-UNCD Surface Chemistry. Among the many bioimmobilization chemistries of chemical vapor deposition (CVD) diamond surfaces,49,50 we use the UV−alkene chemistry due to its ability to withstand hydrolysis and result in better IgG stability.51−53 The UV−alkene chemistry has shown improved temporal stability of antibodies compared to glass surfaces when exposed to saline media at 37 °C for prolonged periods extending up to 2 weeks. An as-obtained 4 in. BD-UNCD wafer was diced into 12 mm × 16 mm chips, cleaned, and functionalized with mouse IgG as shown in Supporting Information Scheme S2 and procedure detailed in the Supporting Information. Hamer’s group has shown that the nonspecific binding of proteins on UNCD can be reduced through PEG immobilization. Therefore, we also examined the use of PEG coimmobilization with mouse IgG. Effect of BD-UNCD Surface Chemistry and DEP on Preconcentration of Beads. Figure 1 shows the microfluidic preconcentrator used in this study. Figure 1A shows the device was constructed of an ITO-coated glass slide, a patterned double-sided tape, and the functionalized BD-UNCD chip. Details of device construction are provided in the Supporting

impedance spectroscopy, or amperometry-based electrical biosensing. Boron-doped diamond is an attractive material to develop such electrodes because it has the widest potential window for aqueous electrolytes, ability to withstand high potentials as well as corrosive chemicals, and results in reduced fouling compared to other electrode materials.40−44 We found that boron-doped ultrananocrystalline diamond (BD-UNCD) films allow application of DEP at bare electrodes as shown in Supporting Information Figure S1. This paper provides preliminary results from our overarching effort to build a novel pathogen detection assay, DEP-enhanced microfluidic impedance biosensor, which uses immunobeads to isolate pathogens from samples, followed by DEP-assisted pathogen capture at BD-UNCD microelectrodes and quantification of the capture using impedance spectroscopy. One of the requirements for using DEP on-chip is the use of nonconductive media, which can sustain the high electric fields required for DEP motion of the pathogens. Often the pathogens from the clinical or the food samples need to be suspended in an isotonic sugar solution or a proprietary formulation to achieve preconcentration.45,46 In our assay, we use immunobeads to isolate the pathogen of interest, thereby reducing interfering agents from the sample and incrementing the selectivity in pathogen sensing. Further, the immunobead acts as a DEP tag during DEP preconcentration as the bead will experience a larger DEP force compared to the bacteria alone. However, nonspecific binding becomes an issue with the use of immunobeads. The purpose of this paper is to report the effect of bead chemistry on the capture efficiency and specificity during pathogen isolation and the effect of bead surface chemistry, BD-UNCD surface chemistry, and DEP on the capture capability and selectivity during immunobeads capture at BD-UNCD.

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METHODS Bead Selection and Its Surface Chemistry. We chose IDC polystyrene beads from Invitrogen, primarily due to it being surfactant-free and its availability in 4 μm diameter with its surface modified with carboxylate, epoxy-/sulfate, or aldehyde-/sulfate functional groups. These are the most widely used functional groups for conjugating antibodies as shown in Supporting Information Scheme S1. The amount of protein loading was characterized by micro-BCA assay and absorption at 280 nm. Coimmobilization of poly(ethylene glycol) (PEG) on immunodiagnostic beads has been shown to reduce nonspecific binding by Yuan et al.47,48 Thus, we also examined the effect of coimmobilizing PEG and antibodies on beads and BD-UNCD. To compare with the state-of-the-art beads, we also tested the epoxy-modified Dynal magnetic beads. Detailed method and calculations for the preparation of the different types of beads are provided in the Supporting Information. All the beads were suspended in 50 mM PBS buffer with 130 μg/ mL sodium azide. The beads were discarded after 1 month of storage at 4 °C. Effect of Bead Selection and Its Surface Chemistry on Pathogen Isolation. Beads functionalized with anti-Escherichia coli O157:H7 IgG with or without PEG (MW 5000) coimmobilization were used to capture live E. coli O157:H7 from isolate cultures containing approximately 1000 E. coli O157:H7 cfu/mL or cocultures containing 500 E. coli O157:H7 cfu/mL and 500 E. coli K12 cfu/mL approximately. The beads were mixed with cultures for 1 h at 20 °C using an orbital shaker in case of polystyrene beads and a HulaMixer for

Figure 1. (A) Exploded view of the microfluidic preconcentrator showing different layers of construction. (B) A packaged microfluidic preconcentrator under testing. B


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Analytical Chemistry Information. Figure 1B shows the device assembled and connected to a DEP voltage source using two minialligator clips with smooth jaws. Prior to assembling, using a diamondtipped pen, the surface of the functionalized BD-UNCD chip was scratched in the middle as shown in Figure 1B. This created two discontinuous areas on the chip: the right half of the chip where DEP was applied and the left half where DEP was not applied. The tubings and microfluidic connectors were blocked with casein. To test the specific and nonspecific adsorption of functionalized bead on functionalized BD-UNCD surface, 1 mL of goat antimouse conjugated beads (105 beads/ mL) was pumped into the channel at 10 μL/min. Effect of DEP was observed while applying a 6 Vp‑p, 40 kHz, square wave to BD-UNCD and grounding the ITO. The channel was then washed with PBS−T20 for 20 min at 100 μL/min and PBS for 20 min at 100 μL/min to remove the nonspecifically bound beads prior to quantification of beads using fluorescence microscopy.

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RESULTS AND DISCUSSION Bead Functionalization with IgG and PEG. Using bovine γ-globulin standards allowed measuring the amount of IgG in solution before and after bead functionalization, and thus predicting the IgG loading on each bead type using optical absorption at 280 nm as well as micro-BCA assay. As shown in the Supporting Information Figure S2, we found that a reaction time of 2 h is adequate for IgG attachment to either type of bead, and epoxy-/sulfate polystyrene beads and epoxy-modified magnetic beads showed higher IgG loading compared to aldehyde-/sulfate and carboxylate-modified polystyrene beads. The IgG loading on the aldehyde-/sulfate and carboxylatemodified polystyrene beads indicated formation of a near monolayer coverage (7.1 μg as calculated in the Supporting Information), while the IgG loading on epoxy-functionalized beads was nearly double the amount of IgG required for a monolayer. This may be explained by the hairy surface of the epoxy-functionalized beads as reported previously,54 compared to the smooth structure of the aldehyde-/sulfate and carboxylate-modified polystyrene beads. The coimmobilization of PEG to IgG-coated beads was confirmed via ζ-potential measurements. As shown in Supporting Information Figure S3, the electrophoretic mobilities of the beads as received, after attachment of IgG and after attachment of PEG were measured. The values for ζpotential as shown in Supporting Information Figure S4 were calculated using the Smoluchowski equation. Since ζ-potential is the potential difference between the dispersion medium and the slipping layer of the particle, we see a change in ζ-potential with the attachment of PEG on the bead surface. Overall, the ζpotential values for all the beads with IgG/PEG coimmobilization were close to −50 mV. Effect of Bead Selection and Its Surface Chemistry on Pathogen Isolation. Figure 2A shows percent capture efficiency (CE%) for tests conducted from PBS containing ∼1000 E. coli O157:H7 cfu/mL. CE% was defined as ([E. coli O157:H7 count captured by beads − E. coli O157:H7 count in negative control]/E. coli O157:H7 count initially present × 100). Supporting Information Table S1 shows two-way analysis of variance (ANOVA) results indicating that the bead chemistry, and not the PEG coimmobilization, affected the CE%. Following paired t tests were used to identify specific differences. We found that the carboxylate-modified beads with IgG/PEG coimmobilization resulted in highest CE% (∼64.8%

Figure 2. Pathogen capture efficiency as measured from spiked PBS using beads functionalized with anti-E. coli O157:H7. Efficiency to capture E. coli O157:H7 from (A) PBS spiked with 1000 cfu/mL target bacteria and (B) PBS spiked with 500 cfu/mL target bacteria and 500 cfu/mL nontarget bacteria E. coli K12. (C) Selectivity percentage in capturing target bacteria. The sample size is three in each trial, and a total of three trials were conducted. Each bead type was tested with PEG coimmobilization (red columns) and without PEG (blue columns). Error bars indicate standard error of mean calculated via error propagation.

± 3.7%). This was found higher (p = 0.005) than the 45.26 ± 2.7 CE% demonstrated by the carboxylate-modified beads without PEG. One would expect that, since epoxy-/sulfateC


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Figure 3. Change in specific and nonspecific binding of beads upon coimmobilization of PEG and IgG on the BD-UNCD surface. Three types of beads, epoxy-/sulfate (red columns), aldehyde-/sulfate (green columns), and carboxylate-modified (blue columns), were used. (A) Specific binding of beads decorated with antimouse IgG on the BD-UNCD surface decorated with mouse IgG with or without PEG. (B) Nonspecific binding of beads decorated with antimouse IgG on the BD-UNCD surface blocked with casein with or without PEG. (C) The specific binding measurements in panel A when performed in the presence of a DEP field. (D) The nonspecific binding measurements in panel B when performed in the presence of a DEP field.

modified polystyrene beads showed a higher IgG loading, it would result in a higher CE%. However, we found that the epoxy-/sulfate-modified polystyrene beads showed the lowest CE%, ∼32.8% ± 8.6% with PEG, and ∼30% ± 8.82% without PEG. This may be due to crowding on the bead surface that results in larger steric hindrance to access the Fab sites for antigen binding. Figure 2B shows the influence of PEG coimmobilization on CE% for tests conducted from PBS containing ∼500 E. coli O157:H7 cfu and ∼500 E. coli K12 cfu. Supporting Information Table S2 shows ANOVA results indicating again that the bead chemistry, and not the PEG coimmobilization, impacted the CE%. Here, we found that the carboxylate-modified beads showed highest CE%. Compared to the capture study from isolate cultures (Figure 3A), the CE% obtained with carboxylate-modified beads with PEG was not statistically different; however, we found a higher CE% for carboxylatemodified beads without PEG when the capture was conducted from mixed culture. The epoxy-/sulfate-modified polystyrene beads gave the lowest CE% among the beads tested. Also compared to the capture study from isolate cultures (Figure 3A), the average CE% obtained with epoxy-/sulfate-modified beads with PEG was higher but statistically insignificant. Similarly for the aldehyde-/sulfate-modified polystyrene beads

and the epoxy-modified magnetic beads, the CE% obtained from isolate and mixed cultures were statistically indifferent. However, we noticed higher standard deviations in the CE% obtained during the capture tests from mixed cultures. Figure 2C shows percent selectivity (S%) for E. coli O157:H7 capture from the cocultures. The S% was defined as ([E. coli O157:H7 count captured by beads − E. coli O157:H7 count in negative controls]/[total bacteria count captured − total bacteria count captured in negative controls] × 100). Supporting Information Table S3 shows ANOVA results indicating statistically insignificant difference in S%. Since the starting concentrations of the bacteria was low (∼500 cfu/mL), we tried to make sure that we count the bacteria by plating major portions of the supernatant (100 μL three times out of the 900 μL supernatant). Any deviation in the number of colony counts got multiplied by a conversion factor (>1) to calculate the cfu/mL. The same also applied to control experiments. Hence, the standard error of the mean calculated to be higher. We believe that if more trials were conducted the error would reduce and significant difference would be seen. Looking at pair t test results in Supporting Information Table S4, it appears that there would be an obvious difference between the carboxylate-modified beads with PEG and the epoxy-/sulfate polystyrene beads with PEG. D


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Analytical Chemistry Table 1. Sp% Values Calculated from the Data in Figure 3

carboxylate-modified beads was the lowest, irrespective of the presence of PEG on BD-UNCD. Also, the application of DEP did not change the way PEG coimmobilization influenced the specific capture as noted earlier in the absence of DEP; statistical indifference in specific capture was found with and without PEG on BD-UNCD (see Supporting Information Tables S7 and S11). Overall, the use of DEP resulted in higher specific capture of all the beads. The use of epoxy-/sulfate beads showed an increase in specific capture of ∼60% with PEG on BD-UNCD and ∼63% without PEG. Carboxylate beads showed an increase in specific capture of ∼19% with PEG on BD-UNCD and ∼43% without PEG. Aldehyde-/ sulfate beads showed an increase in specific capture of ∼37% with PEG on BD-UNCD and ∼50% without PEG. Next, as illustrated in Figure 3D, we tested how immobilization of PEG on BD-UNCD altered the nonspecific binding of beads in the presence of DEP. The BD-UNCD chips were functionalized with PEG and blocked with casein prior to flowing antimouse IgG-coated beads in the presence of DEP. We found that the addition of PEG on BD-UNCD reduced the nonspecific binding of the epoxy-/sulfate beads by ∼37%, which is higher compared to that found in the absence of DEP. The nonspecific binding of aldehyde-/sulfate and carboxylatemodified beads showed statistically insignificant change on adding PEG to the BD-UNCD surface (see Supporting Information Tables S8 and S12). Overall, like the observations on specific binding in Figure 3C, the nonspecific binding of all beads had increased with DEP compared to the nonspecific binding without DEP. To examine closely the extent of DEPmediated increase in specific binding over nonspecific binding, we calculated percent specificity (Sp%) as (bead captured specifically/bead captured specifically and nonspecifically × 100). Table 1 shows the Sp% values calculated from the data in Figure 3 and probability values from a paired t test examining the effect of PEG or DEP. Supporting Information Tables S13−S16 show results of two-way ANOVA. We find that statistically DEP does not affect the Sp% (p > 0.05). Further, the Sp% in experiments without DEP show that the addition of

Overall, the results in Figure 2 show that carboxylatemodified polystyrene beads with PEG give the best CE% when capturing pathogen from isolate or mixed culture. Effect of IgG/PEG Coimmobilization on BD-UNCD. Figure 3 summarizes the effect of coimmobilizing IgG/PEG on BD-UNCD. Supporting Information Tables S5−S8 and S9− S12 summarize the results from two-way ANOVA and paired t tests, respectively. As shown in Figure 3A, BD-UNCD chips were functionalized with mouse IgG, with or without PEG, and further treated with a blocking protein (casein). The functionalized BD-UNCD chips were then packaged with a microfluidic channel on top, and epoxy-/sulfate, aldehyde-/sulfate, or carboxylate-modified beads decorated with antimouse IgG (no PEG) were passed through the microchannel to learn how coimmobilization of PEG and IgG on BD-UNCD affected the bead capture. We did not use magnetic beads as they tend to settle faster in microfluidics. A 4 mm2 area of the microfluidic channel was imaged to enumerate the bead capture. As shown in the two-way ANOVA (Table S5) we find somewhat of an influence of both bead chemistry and PEG on BD-UNCD on the specific capture of beads. There was an increase in specific capture with PEG on BD-UNCD, with the epoxy-/sulfatemodified beads captured the most. Next, as illustrated in Figure 3B, we tested how immobilization of PEG on BD-UNCD affected the nonspecific binding of beads. The BD-UNCD chips were functionalized with PEG and blocked with casein. Tables S6 and S10 show that bead chemistry significantly affects the specific bead capture, while PEG coimmobilization had a negligible effect. Next, we performed the same capture experiment as in Figure 3, parts A and B, while applying DEP between the ITO-coated glass top and the BD-UNCD floor of the microfluidic channel. We wanted to see if the surface chemistry of BD-UNCD was robust to withhold DEP conditions (6 Vp‑p for 100 min); specifically, we looked for changes in the specific and the nonspecific binding possibly due to changes in the IgG, PEG, or blocker protein content on BDUNCD. As shown in Figure 3C, we found that the specific capture of epoxy-/sulfate beads was highest, and that of E


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Figure 4. Change in specific and nonspecific binding of beads upon coimmobilization of PEG and IgG on bead surface. Three types of beads, epoxy-/sulfate (red columns), aldehyde-/sulfate (green columns), and carboxylate-modified (blue columns), were used. (A) Specific binding of beads decorated with antimouse IgG with or without PEG to the BD-UNCD surface decorated with mouse IgG and PEG. (B) Nonspecific binding of beads decorated with antimouse IgG with or without PEG to the BD-UNCD surface blocked with casein and PEG. (C) The specific binding measurements in panel A when performed in the presence of a DEP field. (D) The nonspecific binding measurements in panel B when performed in the presence of a DEP field.

Table 2. Specificity of Capture Calculated from Data in Figure 4

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sulfate and aldehyde-/sulfate beads, but for carboxylatemodified beads the specific capture increased by ∼12%. We noticed a reduction in standard deviation for the specific capture when using PEG on beads. Overall, the use of DEP resulted in higher specific capture of all the beads. The use of epoxy-/sulfate beads showed an increase in specific capture of ∼48% with PEG on beads and ∼60% without PEG. Carboxylate beads showed an increase in specific capture of ∼56% with PEG on BD-UNCD and ∼19% without PEG. The aldehyde-/sulfate beads showed an increase in specific capture of ∼33% with PEG on BD-UNCD and ∼37% without PEG. Next, as illustrated in Figure 4D, we tested how the IgG/PEG coimmobilization on the beads affected their nonspecific binding in the presence of DEP. The BD-UNCD chips were functionalized with PEG and blocked with casein prior to flowing antimouse IgG-coated beads, with or without PEG, in the presence of DEP. Overall, we see a considerable increase in the nonspecific binding of all types of beads due to DEP when compared to Figure 4B. However, as shown in Supporting Information Tables S20 and S24, the addition of PEG produces a statistically significant reduction in nonspecific binding even in the presence of DEP, similar level to that seen in the absence of DEP in Figure 4B. Table 2 shows that the Sp% does not significantly change due to the presence of DEP. Further, in the presence of DEP, the addition of PEG reduces the nonspecific binding of aldehyde-/sulfate and carboxylate-modified beads and not statistically enough for epoxy-/sulfate. The addition of PEG to the beads and BD-UNCD brings the Sp% values of the epoxy-/sulfate and the carboxylate-modified beads statistically close. Supporting Information Tables S25−S28 show two-way ANOVA statistics. We find that the bead chemistry and PEG on beads makes a significant difference on Sp% with or without DEP.

PEG to the BD-UNCD surface increased the Sp% for only the carboxylate-modified beads (p ≤ 0.05). Similarly, the Sp% in experiments with DEP shows that the addition of PEG to BDUNCD increased the Sp% only for the epoxy-/sulfate beads. Overall, the bead chemistry has a major impact in the presence or absence of DEP, but PEG on BD-UNCD only has an impact in the presence of DEP. Effect of IgG/PEG Coimmobilization on Bead and BDUNCD. Figure 4 summarizes the effect of IgG/PEG coimmobilization on bead and BD-UNCD. Supporting Information Tables S17−S20 and S21−S24 summarize the results from two-way ANOVA and paired t tests, respectively. As shown in Figure 4A, the BD-UNCD chips were functionalized with mouse IgG and PEG and further treated with a blocking protein (casein). The functionalized BD-UNCD chips were then packaged with a microfluidic channel on top, and epoxy-/sulfate, aldehyde-/sulfate, or carboxylate-modified beads decorated with antimouse IgG, with or without PEG coimmobilization, were passed through the microchannel. With IgG/PEG coimmobilization on BD-UNCD, we studied how coimmobilization of PEG on the beads affected the bead capture. Like the above studies, a 4 mm2 area in the center of the microfluidic channel was imaged to enumerate bead capture. We found that without PEG on beads, the numbers of epoxy-/sulfate, aldehyde-/sulfate, and carboxylate beads captured were statistically similar (see Supporting Information Tables S17 and S21). After the addition of PEG to the beads, the number of epoxy-/sulfate beads was the highest, followed by the aldehyde-/sulfate beads, and the carboxylate-modified beads were least captured. This difference arose because the addition of PEG to the beads increased the specific capture of epoxy-/sulfate beads by ∼11%, but it did not affect the specific capture of the aldehyde-/sulfate beads and reduced the specificcapture of the carboxylate beads by ∼14%. However, the addition of PEG reduced the standard deviation in capture of all three beads. Next, as illustrated in Figure 4B, we tested how the IgG/PEG coimmobilization on the bead affected the nonspecific binding on BD-UNCD. The BD-UNCD chips were functionalized with PEG, blocked with casein, and packaged with a microfluidic channel prior to flowing beads decorated with antimouse IgG, with or without PEG. We found that the addition of PEG to the beads reduced the nonspecific binding for all three beads, epoxy-/sulfate beads by ∼39%, aldehyde-/sulfate beads by ∼52% and carboxylate beads by ∼25%. See Supporting Information Tables S18 and S22 for statistics. Table 2 lists the calculated Sp% values calculated using data from Figure 4, parts A and B. We see that the Sp% increases due to the addition of PEG on beads, ∼8% for epoxy-/sulfate beads, ∼6% for aldehyde-/sulfate beads, and ∼11% for carboxylate-modified beads. Comparing the results in Figures 3B, and 4B, we find the lowest nonspecific binding is achieved by IgG/PEG coimmobilization on both, beads and BD-UNCD. Figure 4C shows the bead capture count when the experiment in Figure 4A was repeated in the presence of DEP. The BD-UNCD film was functionalized with mouse IgG and PEG and blocked with casein prior to flowing beads decorated with antimouse IgG, with or without PEG, in the presence of a DEP field. Supporting Information Tables S19 and S23 capture the statistics. We found that the specific capture was highest for epoxy-/sulfate beads, irrespective of the presence of PEG on the bead. In the presence of DEP, PEG coimmobilization did not improve specific capture of epoxy-/

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CONCLUSIONS In summary, we show how one could design an immunobeadsbased assay for on-chip DEP preconcentration of rare bacteria at bare BD-UNCD electrodes. In our experiments with polystyrene beads with three different surface chemistry, namely, epoxy-/sulfate, aldehyde-/sulfate, and carboxylatemodified beads, ANOVA shows that bead chemistry plays a significant role in controlling the specific and nonspecific capture during pathogen isolation as well as capture at the BDUNCD electrodes. In an effort to reduce nonspecific binding, a 5K MW PEG was also coimmobilized with IgG on the beads as well as the BD-UNCD electrode. The attachment of PEG on beads did not results in significant changes in specific or nonspecific capture during the pathogen isolation; however, it does alleviate nonspecific binding of the beads to the BDUNCD electrodes. Regardless, the carboxylate-modified beads with PEG result in the highest CE% (65%) and S% (95%). Comparing this performance to state-of-the-art immunomagnetic beads, the carboxylate-modified polystyrene beads coimmobilized with PEG result in similar or better CE% or S %. Using a microfluidic DEP preconcentrator, a 6 V DEP bias (2400 kV/m) was successfully applied for 100 min without damaging the bare BD-UNCD electrodes. DEP application increases the specific as well as nonspecific capture at the BDUNCD electrode; hence, the overall specificity (Sp%) in capture remained unchanged. This shows that BD-UNCD will allow a class of new electrical biosensors to be realized where DEP is directly applied to the electrode in a biosensor. G


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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03227. Source of materials, detailed IgG/PEG immobilization methods on beads and BD-UNCD, method for preparing the microfluidic preconcentrator, Schemes S1 and S2, Figures S1−S4, and Tables S1−S28 that are referenced within the main text (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 318 257 5112. Fax: +1 318 257 5104. Author Contributions

The manuscript was written through contributions of all authors. W.Z. and A.D.R. designed the experiments, W.Z. conducted the experiments and collected the data, W.Z. and A.D.R. analyzed the data and prepared this manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Research reported in this publication was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under Grant No. P20GM103424. We are thankful to the staff at the Institute for Micromanufacturing and the Center for Biomedical Engineering and Rehabilitation Science at Louisiana Tech University. We are grateful to Drs. R. Bashir (Illinois) and A. Bhunia (Purdue) for sharing E. coli cultures and to Dr. R. Hamers (Wisconsin−Madison) for sharing the UV−alkene chemistry process and chemicals. We are thankful to Drs. K. Macaluso, R. Subramaniam, and K. Kousoulas at the Louisiana State University Veterinary School of Medicine for their guidance on the project. We are thankful to Dr. N. Crews for access to the Dremel drill setup and Dr. J. Pojman for their polymer chemistry for microfluidic interconnects.

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REFERENCES

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Toward a Boron-Doped Ultrananocrystalline Diamond Electrode

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