An ultrahigh temperature flowthrough capillary device

2804 Kyle W. Hukari Kamlesh D. Patel Ronald F. Renzi Jay A. A. West Microfluidics Research Group, Sandia National Laboratories, Livermore, CA, USA

Received May 11, 2010 Revised May 15, 2010 Accepted May 16, 2010

Electrophoresis 2010, 31, 2804–2812

Research Article

An ultra-high temperature flow-through capillary device for bacterial spore lysis Rapid and specific characterization of bacterial endospores is dependent on the ability to rupture the cell wall to enable analysis of the intracellular components. In particular, bacterial spores from the bacillus genus are inherently robust and very difficult to lyze or solubilize. Standard protocols for spore inactivation include chemical treatment, sonication, pressure, and thermal lysis. Although these protocols are effective for the inactivation of these agents, they are less well suited for sample preparation for analysis using proteomic and genomic approaches. To overcome this difficulty, we have designed a simple capillary device to perform thermal lysis of bacterial spores. Using this device, we were able to super heat (1951C) an ethylene glycol lysis buffer to perform rapid flowthrough rupture and solubilization of bacterial endospores. We demonstrated that the lysates from this preparation method are compatible with CGE as well as DNA amplification analysis. We further demonstrated the flow-through lysing device could be directly coupled to a miniaturized electrophoresis instrument for integrated sample preparation and analysis. In this arrangement, we were enabled to perform sample lysis, fluorescent dye labeling, and protein electrophoresis analysis of bacterial spores in less than 10 min. The described sample preparation device is rapid, simple, inexpensive, and easily integratable with various microfluidic devices. Keywords: Ethylene glycol / Microfluidics / PCR / Protein electrophoresis / Sample preparation DOI 10.1002/elps.201000176

1 Introduction Field portable analysis of biological agents is critically dependent on the ability to rapidly prepare samples for analysis. An important step in sample preparation is the lysis and solubilization of the agent for analysis. This step is complicated by the wide variability in the stability of biological agents to lysis and solubilization [1]. As a result, a variety of techniques have been developed for the lysis of viruses and bacterial agents, including chemical and detergent lysis [2, 3], enzyme treatment [4], sonication [5], heating [6], and glass bead milling [7]. Bacterial spores, in particular, are extremely resistant to lysis and solubilization, and often require a combination of these aforementioned techniques to prepare them for analysis [8, 9]. Many of these lysis techniques complicate subsequent analysis due to addition of chemical additives or enzymes to the samples, which can interfere with the amplification, labeling, or analytical analysis [6].

Correspondence: Dr. Jay A. A. West, Arcxis Biotechnologies, 6920 Koll Center Parkway, Pleasanton, CA 94566, USA E-mail: [email protected]; [email protected] Fax: 11-925-261-7901

Abbreviation: EG, ethylene glycol

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The goal of this study was to develop a rapid sample processing technique that could be used with a variety of analytical techniques including capillary-based protein electrophoresis and PCR-based DNA analysis. In addition, we aimed to develop a protocol able to process extremely robust agents for direct integrated analysis using microfluidic devices. Several sample processing techniques have been developed for use and integration with microfluidic devices. These techniques and microfluidic devices include integrated detergent mediated lysis [10, 11], laser-mediated cell lysing [12], electric field-mediated lysis [13–15], or the combination of these techniques [16]. Furthermore, highly integrated systems have been developed that allow for the lysis, concentration, purification, and analysis of DNA from Escherichia coli [17–19]. The majority of these studies performed sample processing on relatively labile eukaryotic cell types and bacteria. However, comparatively few studies have been focused on the development of processes for the rapid lysis and analysis of bacterial spores, which can be easily integrated with microfluidic analysis platforms. Belgrader et al. previously demonstrated that sonication of Bacillus subtilis spores in the presence of silica beads was an effective technique for lysis prior to PCR analysis of spore DNA [20]. Taylor et al. followed up these studies by demonstrating real-time PCR analysis using a similar device [21]. Although these methods are effective at disrupting the spore www.electrophoresis-journal.com


coat for analysis of internal DNA, they do not effectively solubilize proteins, which is required for subsequent protein fingerprinting, a technique which has been developed for rapid identification of bacteria [22–24] and viruses [25]. We devised a scheme to employ ultra high-temperature solubilization for bacterial spores to enable the rapid preparation of biological agents for analysis either by protein electrophoresis or by PCR. We then demonstrated the lysates from these preparations were compatible with CGE analysis of proteins with microchip-based laser-induced fluorescence detection. We further demonstrate that the eluants containing DNA generated from the capillary lyser were compatible with PCR. This apparatus was then integrated with a series of syringe pumps to enable automated fluorescent dye labeling and sample buffer mixing with the lysates of the bacterial agents. Using this system, complete solubilization, labeling, and analysis of Bacillus atrophaeus, Bacillus anthracis, Bacillus cereus, and B. subtilis spores were possible. We found that we were able to directly couple this sample preparation device to a hand-portable analysis platform. The device and developed process provide the ability to rapidly prepare samples of bacterial spores for either protein or DNA analysis from the same sample without requiring additional chemical or mechanical sample preparation steps.

2 Materials and methods 2.1 Reagents Fluorescent dyes 8-hydroxypyrene-1,3,6-trisulfonic acid and fluorescamine dyes were purchased from Molecular Probes (Eugene, OR). Ethylene glycol (EG), boric acid, SDS, a-lactalbumin (14.4 kDa), carbonic anhydrase (32.5 kDa), ovalbumin (45 kDa), and BSA (66 kDa) were purchased from Sigma (St. Louis, MO). Cholecystikinin flanking peptide is a synthetic peptide (1.1 kDa) obtained from Commonwealth Biotechnologies (Richmond, VA) and used as purchased. Polyethylene glycol/polyethylene oxide sieving gel, in the 14–200 kDa range (part & sharp 477416), was purchased from Beckman Coulter (Fullerton, CA). All other chemicals purchased were reagent grade or better and were used without any further purification.

2.2 Bacterial growth and spore production Bacillus atrophaeus, B. cereus, and B. subtilis spore preparations were purchased (Raven Biological, Omaha, NB). In-house preparation of B. anthracis (Delta Sterne) was performed as previously described by Nicholson and Setlow [26]. Bacillus anthracis (Delta Sterne) strains are nonencapsulated, nontoxigenic strains of B. anthracis and were used in a BSL 2 laboratory. Briefly, initial growth for cultures took place in Luria-Bertani media (20 g/L, LB Broth, Difco). Small-scale cultures incubated overnight at 371C were diluted and used to inoculate large-scale cultures at an od of 0.05 at 600 nm. & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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When cultures reached an od between 0.4 and 0.6 at 600 nm, cells were spun down for 10 min at 5000 g and resuspended in an equal volume of sporulation media. Sporulation media consisted of 1.0 mL of 0.1 M CaCl2, 4.0 mL of 5% L-glutamate (pH 7.0 with 10 M NaOH, filter sterilized), 4.0 mL of 1 M MgSO4 7H2O, and 90 mL of sporulation salts (3.3 mM FeCl3 6H2O, 40.1 mM MgCl2 6H2O, 100 mM MnCl2 4H2O, 0.01 M NH4Cl, 75 mM Na2SO4, 50 mM KH2PO4, 1.21 103 M NH4NO3) per 100 mL [26, 27]. Cultures remained incubated at 371C for 48 h. Samples were then centrifuged at 10 000 g and the supernatant decanted. All spore preparations either purchased or grown in house were purified prior to use in the lysing experiments. Purification included three washes with sterile-distilled DI H2O, one wash with 1 M KCl/0.5 M NaCl, one wash with 1 M NaCl, one wash with 2 M NaCl, one wash with 0.25% SDS, and three washes with sterile-distilled DI H2O to remove extraneous vegetative cell lysates. All washes occurred in one-fourth of the original culture volume with centrifugation at 10 000 g for 10 min. Purified spores were then resuspended in steriledistilled/DI H2O and stored at 41C.

2.3 Flow-through lysis of bacterial spores A flow-through lysing apparatus was constructed by wrapping a 32-gauge uncoated copper wire (Arcor, Northbrook, IL) around a 10 cm long 550/640 mm id/od fusedsilica capillary (Polymicro Technology, Phoenix AZ) with approximately 20 windings/cm as shown in Fig. 1. The external windings were then soldered to a simple two-prong connector for operation using a standard 10 V DC power supply (Hewlett Packard, Palo Alto, CA). Current passing through the windings resistively heated the capillary tube. A second capillary (250 id/360 od) was then inserted into the larger heater capillary. The second capillary served as a removable and replicable fluid conduit through which the sample could be passed. Care was taken to reduce airflow over the device to stabilize the temperature of the heating element by encasing the device in a draft shield. A range of temperatures from room temperature to 1951C could be achieved by adjusting the power supply voltage. The voltage applied and current from the power supply were monitored to ensure reproducible performance. The internal temperature of the flow-through capillary inside the heater capillary was measured by inserting a thin thermocouple wire (0.06 in) (Omega, Stamford, CT) and reading the values on a hand-held readout monitor (Fluka, St. Louis, MO). Temperature was measured inside the capillary with a flowing stream of EG buffer at the appropriate flow rates to accurately calibrate the temperature of the working system.

2.4 Sample preparation Bacterial spore samples were pelleted by centrifugation and resuspended in a buffer containing 5 mM boric acid www.electrophoresis-journal.com

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2.5 Microchip CGE

Figure 1. Fabrication of a simple thermal lysing apparatus. A heating element was constructed using a 400/650 mm id/od fused-silica capillary was wrapped with a resistive 32 gauge copper wire (A). A second fused-silica capillary (150/360 mm id/od) was then inserted into the element to allow heating of the lysis buffer. Using this setup, solutions of EG can be passed through the device to heat samples to as high as 1951C. The terminus of the copper wire was then connected to a standard 10 V power supply using a simple solder connection (B).

and 5 mM SDS dissolved in 98% EG. Samples were then transferred to a glass syringe connected to the 250/360 id/od capillary used to deliver sample to the thermal lyser via a syringe pump (Cole Parmer, Vernon Hills, IL). Realtime imaging of the cell lysis for bacterial spores was performed using an inverted light microscope setup to observe both inlet and outlet capillaries. Spore lysis was confirmed by counting spore particles imaged between a glass slide and a cover slip. To quantify the number of spores in each experimental group, a minimum of four images were captured and the particles were counted in each image and averaged. These values were used to determine the amount of spore reduction during the lysing step. Analysis of the samples prepared with the capillary thermal lyser was either performed by manually injecting collected samples onto the miniaturized detection platform or by connecting the lyser apparatus directly to the injection port of the miniaturized electrophoresis platform. In either case, the spores were initially prepared by centrifuging and resuspending in the lysis buffer. During the initial experiments, the heated spore solution was manually labeled by adding a 10 mM fluorescamine (1 mM final concentration) which was then vortexed to mix the sample and the dye. & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Separations were performed on an in-house-fabricated 2.0-cm 2.0-cm fused-silica microfluidic chip, which was fabricated from Corning 7980 fused-silica wafers (SensorPrep, Elburn, IL) using standard wet-etch procedures as described previously [28]. These chips contained a 10-cm separation channel filled with a polyethylene oxide, polyethylene glycol, and EG gel mixture containing SDS (Beckman Coulter) for protein analysis. To prepare the chip for analysis, all channels were flushed with filtered distilled DI water, then evacuated to dryness. This process was repeated three times, after which the chip was then filled with the sieving gel using a gas-tight, 250-mL syringe connected to a flush port. Channels were filled by applying modest pressure (110 cm/Hg) to the syringe for approximately 1–3 min. Buffers were contained in reservoirs connected to the manifold by a capillary-septum interface as described previously [28]. The sample waste, buffer, and waste reservoirs were each filled with approximately 0.75 mL of sieving gel. The sample reservoir was filled with approximately 0.2 mL of sample buffer (5-mM boric acid and 5-mM SDS titrated with 1.0 M NaOH to pH 8.8). The liquid reservoir cartridge was installed on the fluidic manifold, and the electrode plate was connected to the top of the fluidic reservoir cartridge. The injection currents/voltages for optimal injection and separation were programmed using the user interface on the device. Samples were injected electrophoretically onto the separation media in the microchannel as described previously [29, 30], with subtle alterations [28, 31]. Control of the sample plug on-chip was assisted by maintaining the current on the buffer leg between 0.2 and 0.6 mA. Separations were carried out using a constant current of 11.0 mA (at 450 V/cm) on the waste leg. Sample and sample-waste voltages were chosen to reduce sample carryover and prevent sample leakage into the separation channel during a run. Laser-induced fluorescence detection was performed using the epifluorescent (EX 405 nm/EM 475 nm) optics module described previously [25]. To view and analyze the generated electropherograms, the instrument was connected to an external laptop computer.

2.6 DNA amplification PCR analysis was performed using the Ready-To-GoTM RAPID analysis kit from Amersham Biosciences (Sunnyvale, CA). Reactions were carried out using the lysate that was initially diluted 1:16. PCR was performed on this sample along with a series of dilutions. The PCR assay was carried out using two primers; (50 -d[GTTTCGCTCC]-30 ) and (50 -d[AAGAGCCCGT]-30 ) using thermocycling steps as described. Reactions were transferred to a prewarmed thermocycler and incubated at 421C for 15–30 min. The reaction was heated to 951C for 5 min to completely www.electrophoresis-journal.com



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denature the template. Reactions were then cycled 20–45 times depending on the abundance of the target. Following these steps, standard thermocycling steps were executed, including denaturation at 951C for 1 min, 551C for 1 min, and extension at 721C for 1 min. Products of the random primed PCRs were visualized on a 2% agarose E-Gel from InvitrogenTM.

syringe pump for the addition of fluorescamine protein labeling dye. The flow rate ratio between the two syringe pumps was set to achieve a 50% lyzed sample/50% fluorescamine dye solution. This solution was then pumped directly into the hand-portable microchannel electrophoresis instrument described previously to perform microchannel gel electrophoresis of the prepared sample.

2.7 Lyser integration with CE microchip

3 Results and discussion

To determine the best method for the integration of the spore lysing apparatus to the microchip electrophoresis instrument, we conducted several experiments to determine the optimal labeling protocol and buffer system. We previously reported the use of fluorescamine to rapidly label protein as high as 10 mg in a 180 mL solution of 5 mM Boric acid/5 mM SDS pH 8.5 buffer and later adding 20 mL 10 mM fluorescamine dye [25, 28, 31]. To determine the ability to use an alternative buffer for the sample introduction on the microfluidic electrophoresis chip, samples containing 10 mg of protein were dissolved in 180 mL of buffer that contained 5 mM Boric acid/5 mM SDS pH 8.5 buffer in a approximately 98% EG which then mixed with 20 mL 10 mM fluorescamine dye. These samples were then analyzed by electrophoresis using an integrated arrangement of syringe pumps and the electrophoresis system. Because the results of the experiments showed differences in peak heights, we then determine the optimal labeling reaction conditions using a Perkin Elmer dual cuvette fluorimeter (Waltham, MA). The kinetics of this reaction were determined by observing the fluorescent product generation as a function of time (over 360 s). In order to reproducibly observe the increases in fluorescence, a higher volume sample was necessary to detect the product formation. The first reaction was the carried out in 280 mL of standard aqueous buffer with 10 mg of BSA in 10 mL of water, labeled with 20 mL of 10 mM fluorescamine in acetonitrile. The following reactions were an approximation of the low volume labeling done on the capillary apparatus, which is at a 1:1 ratio of lysate to labeling dye. We compared the labeling between standard aqueous to EG buffers. The second reaction contained 150 mL of aqueous buffer mixed with 150 mL of 10 mM flourescamine in ACN, and 10 mg of BSA in 10 mL of DI water. The third reaction was carried out in the same manner except with 150 mL of EG buffer (instead of Aqueous buffer), 150 mL of 10 mM flourescamine in ACN, and 10 mg of BSA in 10 mL of water. At the conclusion of each of the kinetics experiments, each sample was then fluorometrically scanned (600 s after the addition of the fluorescamine) between 425 and 545 nm to confirm that the fluorescent protein formation was detected at the correct emission wavelength. These experiments enabled the ability to integrate the lyser and dye introduction with the microfluidic electrophoresis chip. In the integrated arrangement, the lyser device was connected to the electrophoresis platform in conjunction with a second

This study focused on the development of a rapid, universal sample preparation protocol, and device for the lysis and solubilization of biological agents such as bacterial spores. We designed and fabricated a simple and inexpensive flowthrough capillary heating device (Fig. 1). The compact device (10 cm) was fabricated using two capillaries. To create the heating element (Fig. 1A), the first capillary (400/650 id/od) was wrapped on the exterior with a high resistance copper outer wire (0.5 mm). The second inner capillary (150/350 id/od) was then inserted into the heating element to allow heating of discrete sample flows. In this arrangement, the inner capillary can be exchanged easily to allow the use of multiple capillaries in a single apparatus. The fabrication of these devices is quite simple, requiring only 10 min to assemble the entire system. This device was then mounted on a miniaturized breadboard plate with the heating coil attached to an external power supply via a simple soldered connection (Fig. 1B). The device is routinely capable of rapidly heating solutions, such as EG, to temperatures as high as 1951C (Fig. 2). The thin-wire thermocouple inserted inside the flowthrough capillary was used to measure the internal temperature of the solution. The heated solutions reached a maximum temperature (Fig. 2A) in the device within 0.5 cm (Fig. 1) of the end of the heater tube, and the temperature remained constant between 1 and 5 cm. Figure 2B shows the linear relationship between the temperature and the applied voltage and current. This linearity allowed us to accurately correlate the internal temperature with either applied voltage or current to characterize each lyser device prior to its use for lysing biological agents. We found flow rates as high as 30 mL/min did not appreciably affect internal temperature of the lysing device. Typical flow rates used for bacterial spore lysis were 3–10 mL/min. A compelling finding in these studies was that the columns 10.0 cm long (with a 5 cm heating region) are adequate to achieve the necessary residence time for bacterial spore solubilization as shown in Fig. 3. We found that flow rate of 3.3 mL/min provided adequate residence time (45 s) for spore solubilization. To determine the amount of bacterial spore lysis, preparations of the cells were imaged using phase contrast microscopy. Prior to lysis, the bacterial spores appear evenly dispersed in the solution with a circular structure (Fig. 3A). A standard approach for the lysis of labile cells typically includes the heating of preparations cells to 95–1001C. In our studies, we found that


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Figure 3. Thermal lysis of B. subtilis spores. Solutions of B. subtilis spores were resuspended in an EG-based buffer (A). These solutions were then heated to 1001C (B),1751C (C), and 1951C (D). At a temperature of 1001C, no decrease in spore count was apparent (B). In contrast, temperatures of 1751C produced spore particles that appeared to clump together, and partially lyze (C). At the highest temperature tested, 1951C, this clumping was less apparent and the numbers of spore particles appeared to be dramatically reduced (D).

Figure 2. Determination of temperature within the heating capillary. Internal temperature of the lysing capillary was measured using a thin wire thermocouple inserted into the internal fluidic conduit. Fluid temperature was measured at different locations (A) within the capillary and was also measured to determine the functional relationship between the temperature and the applied voltage or current (B).

bacterial spores heated to 1001C, compared with control (Fig. 3A), that no significant reduction in spore count was discernable, as judged by visual observation (Fig. 3B). In contrast, when the temperature of the internal solution passing through the capillary lyser was increased to 1751C, the spores appeared to reduce in numbers, coagulate, and form clusters (Fig. 3C). A true and representative calculation of percent decrease of particles from these images was difficult due to this clumping effect. Since the lysates of the spore solution were imaged at room temperature, this coagulation of the spores likely occurs from the release of intracellular DNA. These cells with released extracellular DNA presumably formed hybridized structures to bind the partially solubilized spores together as the solution cools during the imaging procedure. This coagulation effect was less pronounced when the lysis temperature was raised to 1951C. Processing spores at this temperature resulted in a dramatic decrease in observable spore particles (Fig. 3D). Counting these spores subjected to this treatment revealed greater than an 80% decrease in the observable B. subtilis spores. & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The lysates from these preparations were further analyzed for protein content and DNA Initially, samples were manually injected onto a microchip-based CE device, mChemLab [25, 28, 31] for protein electrophoresis analysis, or mixed with random primers to perform DNA amplification experiments. When the lysates were used for protein CE analysis, we found that preparation of spores at temperatures below 1751C (Fig. 4A) did not result in significant spore solubilization and subsequently did not produce any detectable proteins in the electropherogram. However, when the conditions for spore lysis were met, the detection of a protein electropherogram of B. anthracis (Delta Sterne) spores was easily measured (Fig. 4A). The data indicated that the rapid lysis and solubilization of robust spores for the generation of protein electropherograms were achievable. We found that the only requirement for the preparation and analysis of a variety of species was optimal temperature selection. To confirm the release and integrity of the inner contents, we isolated and measured the genomic DNA of these spores. We found that prior to the lysis procedure DNA was undetectable. In contrast, after passing the spores through the thermal lyser, recoverable DNA concentrations of 10–100 ng/mL after purification in spore lysates were detected. Using random primers, PCRs of B. anthracis (Delta Sterne) lysate dilutions were first performed to determine the amount of lysate needed to obtain products and also to visualize the banding pattern produced by each set of random primers. Our best results were obtained when we eliminated the DNA purification step prior to the PCR. We did find, however, that EG inhibits the PCR when present at high concentrations. We performed several experiments to www.electrophoresis-journal.com


determine the concentration of EG that inhibited the PCR. Subsequently, the EG concentrations present in the dilutions were maintained to keep the solutions r4%. Similar to the protein CE analysis, products from the PCRs could not be visualized on the electrophoresis gels from unlyzed spores (Fig. 4B). In contrast, when lysis and solubilization conditions were met, products from the PCRs were readily detectable on the gels. Furthermore, we were able to obtain products from all dilutions (2–2000) of the concentrated lysate. Using this process, we were able to generate PCR products from dilute spore solution concentrations, as low as 295 spores in 50 mL as seen in the 1:2000 dilution in Fig. 4B. Our overall goal in these experiments was to develop a field-portable sample preparation tool for the analysis of biological agents. To achieve this capability, we coupled the flow-through thermal lyser to a second syringe pump to facilitate automated sample preparation and fluorescent dye labeling. The lyser and pump system was then integrated to the mChemlab system for analysis as shown in Fig. 5. To determine the optimal mixing conditions for this process, we first used protein standards to define the operational parameters. Interestingly, during this process we noted an increase in the peak heights of the labeled proteins using EG buffer compared with the previously published [31] protocol preparing the samples in small centrifuge vials (Fig. 6). To determine the mechanism for this increase in peak intensity, we performed several time-course reactions to compare the bench-top fluorescent dye labeling process versus the integrated approach. We first tested the ability to label proteins in a solution consisting of EG. To perform these experiments, an additional capillary and pump were connected to the stream exiting the lyser apparatus. We were surprised to determine that the dye labeling reaction appeared to result in peaks of greater intensity with proteins of higher molecular weight, such as BSA (66 kDa) and b-galactosidase (116 kDa) (Fig. 6A). Although the concentration of dye was significantly higher with this arrangement, we had previously performed the protein labeling step in a large excess of dye [25, 28, 31], and did not expect the kinetics to differ under these conditions. To investigate the mechanism behind this increase in signal-to-noise ratio of the detected peaks, we evaluated the labeling efficiency of fluorescamine, a dye which reacts rapidly with proteins [32], in either an aqueous or the EG-based buffers. These experiments were designed to compare the efficiency and kinetics of the labeling reaction in both the standard aqueous buffer and the EG solution. We have found that the fluorescamine-labeling reagent is effective due to its highly fluorogenic properties and rapid reaction kinetics [28]. Our initial experiments were performed by mixing the dye (dissolved in ACN) into the boric acid and SDS water-based buffer to label BSA. During these experiments, we observed the formation of a precipitate in the aqueous buffer solution used to label BSA protein immediately after the dye was introduced. The formation of this & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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Figure 4. Protein and DNA analysis of B. anthracis (Delta Sterne) lysate eluents. (A) Preparations of both unlyzed and lyzed spores were analyzed using CE. Our results demonstrate in contrast to unlyzed spores CE analysis of lysates of solubilized spores generate an observable protein electropherogram. In a separate set of experiments (B) unpurified DNA from both unlyzed and lyzed B. anthracis spores was amplified using random primers. (Lane 1) 100 bp DNA ladder. (Lane 2) DNA amplification was performed from on lysate generated from spores lyzed at high (1012/mL) concentration. To test the sensitivity of this reaction, we also amplified DNA from low spore concentration lysate. (Lane 3) We found that we were able to perform successful PCR on solutions as dilute as 6 spores/mL. (Lane 4) The reaction was specific to the internal contents of the spores, as unlyzed spores did not generate any PCR products.

precipitate was transient and appeared to dissolve back into solution in approximately 1 min. This reaction was then monitored using a fluorimeter, where a dramatic but transient increase in fluorescence was measured, which www.electrophoresis-journal.com

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Figure 5. Integration of the sample preparation and analysis platform. The flow-through lyser device was integrated to an additional syringe pump to supply fluorescamine dye (in ACN) to the lysate for direct injection into a hand-portable (mChemLab) electrophoresis instrument.

decreased back to baseline after approximately 1 min (Fig. 6B, H2O std). This transient increase and subsequent decrease in fluorescence was followed by a steady increase in fluorescence intensity over a period of 6 min, at which time the reaction appeared to have reached a near-maximum fluorescence level (Fig. 6B, H2O std). In another similar experiment, we performed the labeling of protein with a higher relative concentration of the fluorescamine dye (Fig. 6B, H2O fast). During this experiment, a similar pattern was observed; an immediate dramatic increase in fluorescence followed by a decrease back to baseline, followed by a gradual increase over approximately 6 min. Compared with the first reaction, this reaction generated a greater amount of fluorescence over time, which we attribute to a greater amount of protein labeling. In the third experiment, we tested the same BSA–fluorescamine reaction in the EG lysis solution (Fig. 6B, EG buffer). In these samples, we did not observe a transient precipitant formation or a transient increase in fluorescence. Rather, a steady increase in fluorescence appeared over a period of 180–200 s, where the fluorescence intensity was near maximal. Compared with the first two reactions, the labeling reaction carried out in EG appeared to generate approximately a maximal sevenfold increase in fluorescent product at 180 s. We also measured the fluorescence spectrum at approximately 10 min after the addition of fluorescamine, and found that the EG solution remained roughly threefold higher in the observed fluorescence intensity. Taken together with the electropherograms, these results indicated that a greater amount of specific BSA–fluorescamine labeling appeared to be generated using the EG lysis solution. These results are similar to those found by Udenfriend et al. [32] where they found rapid dye labeling of proteins was possible in organic-based solutions. Using the integrated system, we then tested the ability to complete the entire sample preparation and perform electrophoretic separations of a variety of bacterial spores. Briefly, as in the initial lysis experiments, samples were diluted into the EG lysis solution then drawn into a gas-tight syringe. The loaded samples were then injected through the flowthrough lysing apparatus and were subsequently labeled with fluorescamine dye and injected onto the miniaturized elec& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 6. Comparison of labeling efficiency between EG buffers and standard aqueous buffers. The labeling of protein standards (A) in the EG buffer appeared to be more efficient for the labeling of larger protein species as judged by CGE. (B and C) The kinetics of fluorescamine dye labeling of BSA was measured. In contrast to dye labeling reactions carried out in aqueous buffers, the reaction in the EG buffer produced a higher level of fluorescence in a shorter time period (B), reaching near maximal signal in 180 s, where as reactions in the aqueous buffer had not reached maximal fluorescence in 360 s. The fluorescence spectra for each sample were then collected 10 mins after dye introduction (C). The spectra show that the dye labeling reaction BSA carried out in the EG buffer generated approximately 60 fluorescence units. This was minimum threefold improvement in the total fluorescent products, compared with either of the protocol using the aqueous buffer system.

trophoresis system. Using this system, we performed analysis of a variety of Bacilli spores (Fig. 7). We found that the device was routinely capable of performing lysis and analysis of four www.electrophoresis-journal.com


different spore strains. Analysis of the lysates of B. atrophaeus and B. subtilis (Figs. 7A and B) revealed a single large peak of very high molecular weight. In contrast, analysis of B. cereus and B. anthracis generated several low-molecular-weight proteins in addition to the high-molecular-weight peak that was present. We were surprised by the variability in the generated spore electropherograms. Of particular note was the detection of a large molecular weight peak with a migration time later than our greatest standard BSAIII, which corresponds to a protein of approximately 240 kDa. The peak is Gaussian, and in contrast to the control (grey line in Fig. 7A–D) is present in each of the thermally lyzed samples. We are certain this peak is specific to the spore form of the bacteria, as the ultra high-temperature lysis of the corresponding vegetative cells of these spores does not generate this late-eluting peak (data not shown). We also noted that the electropherograms were all at least subtly different from each other. The differences between the

Figure 7. Integrated lysis and analysis of B. atrophaeus, B. subtilis, B. cereus, and B. anthracis. Using this system, we performed analysis of a variety of Bacilli spores. We found that the device was routinely capable of performing lysis and analysis of four different spore strains including (A) B. atrophaeus, (B) B. subtilis, (C) B. cereus, and (D) B. anthracis. Protein standards (bottom) were used to indicate the respective molecular weight of detected peak.



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B. atrophaeus and B. subtilis strains versus the B. cereus and B. anthracis strains employed may be due to differences in the exosporium coat of the spores [33, 34]. Bacillus subtilis strains including B. subtilis niger (alternatively named B. atrophaeus or B. globigii) are believed to lack a significant spore coat, and, as a result, are more hydrophobic in nature. In contrast, the B. cereus and B. anthracis contain a distinct exosporium coat [33, 34]. It is possible the variability we have seen in our experiments is partly due to these differences. Additional developments on the ultra high-temperature flow-through technique will focus on the identification of the peaks detected in these experiments.

4 Concluding remarks Our goal in these studies was to develop a simple process and device to enable rapid lysis of resistant bacterial spores for subsequent protein labeling and electrophoresis analysis as well as DNA amplification using the PCR. We found the developed lysis method directly coupled with a portable electrophoresis platform could detect the presence of proteins from lyzed spore particles. A significant finding in our studies was the observation that protein dye labeling using the rapid reacting fluorescamine dye was optimal using the EG-based buffers as compared with the aqueous buffers. We found, similar to the previous study [32], that the labeling kinetics were roughly 2.5–4 times more rapid using the EG buffer than the water-based buffer. This was critical to achieving the ability to integrate the lysis of the bacterial spores to the downstream analysis of the soluble proteins. Although the use of EG is a good step forward for integrated analysis using the electrophoresis system, it was not without challenges. One issue we continually encountered was the apparent interference of the EG with the purification and subsequent analysis of both proteins and DNA in these samples using conventional techniques. We remain uncertain of the mechanism of this interference. Although the increased boiling temperature of EG afforded the ability to super-heat these solutions and subsequently lyze the bacterial spores, it is clear that further optimization is required. Such optimization will include testing alternative high-boiling-point solvents that may have less interaction or interference with alternative downstream detection techniques. We also are pursuing the optimization of the current system for the integration of microfluidic devices with real-time PCR assays, and immunological reagent-based detection assays. The authors of this study acknowledge the following individuals for their assistance with these studies. The authors thank Dr. Todd Lane and Richard Gant for the preparation of the bacterial spores. Tom Raber assisted the authors in the fabrication of the thermal lyser. Brad Aubuchan provided engineering support on the mChemlab system for the microchip-based capillary electrophoresis analysis. Finally, the authors thank John Brazzle for the fabrication of the microfluidic chips used in the www.electrophoresis-journal.com

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analysis of the thermal lysates. This study was generously funded by grants from the Department of Defense and the Department of Homeland security, under the Chemical and Biological Countermeasures Program. The authors have declared no conflict of interest.

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