Amperometric Detection of Bacillus anthracis Spores : A Portable , Low-Cost Approach to the ELISA

Antibody-based detection assays are generally robust, a desirable characteristic for in-the-field use. However, to quantify the colorimetric or fluorescent signal, these assays require expensive and fragile instruments which are ill-suited to in-the-field use. Lateral flow devices (LFDs) circumvent these barriers to portability but suffer from poor sensitivity and subjective interpretation. Here, an antibody-based method for detecting Bacillus anthracis spores via amperometric signal generation is compared to ELISA and LFDs.This amperometric immunoassay uses antibody conjugated tomagnetic beads and glucose oxidase (GOX) alongwith the electron mediator 2, 6-dichlorophenolindophenol (DCPIP) for production of a measurable current from a 0.4V bias voltage. With similar sensitivity to ELISA, the assay can be completed in about 75 minutes while being completely powered and operated from a laptop computer. Immunoassay amperometry holds promise for bringing low-cost, quantitative detection of hazardous agents to the field.


Introduction
Anthrax spores are a dormant viable offspring derived from the Gram-positive, rod-shaped bacterium Bacillus anthracis.They are responsible for 3 well-known disease states: cutaneous, gastrointestinal, and inhalation anthrax, the latter of which is characterized by a high mortality rate [1].Due to spore stability and high mortality rate, anthrax spores are ranked high among the potential weapons of bioterrorists [2,3].Hence, a tremendous interest exists for developing quick, reliable, and field-deployable detection devices.DNAbased assays typically have the best sensitivity and specificity but are not readily suited for in-the-field use due to lack of portability and being highly sensitive to environmental contaminants [4].Notably, during the investigation of the USA anthrax attack of 2001, a total of 4,639 real-time PCR reactions were performed and all were negative [5].Positive results were only obtained after culture enrichment when environmental contaminants (dust, debris, and collection buffer surfactants) were eliminated.Alternatively, antibodybased assays are more appealing for in-the-field use due to their environmental robustness and ease of use.
The typical ELISA generates quantifiable results and is fairly sensitive (10 4 cfu/mL) [6] but is too cumbersome and time-consuming to be performed entirely in the field.In addition, expensive and power-demanding instrumentation is needed.Lateral flow devices (LFDs, a.k.a.immunostrips) are a portable and low-cost immunoassay that can be completed in just 15 minutes.However, they are subjectively interpreted and typically characterized by poor sensitivity and frequent false positives.The lower limit for detecting B. anthracis spores is typically around 10 5 to as much as 10 7 cfu/mL depending on manufacturer and/or spore strain and preparation method [7,8].Ideally, for an immunoassay to meet the demands of hazardous agent detection in the field, it would have the sensitivity and dynamic range of an ELISA and the simplicity and quickness of the LFD.
Among the emerging technologies which are advancing the immunoassay state of the art [9], electrochemical signal generation methods are appealing for high sensitivity, low power, and relatively simple instrumentation, along with other advantages [10].For the sake of portability and inthe-field use, the advantages of electrochemical detection are quite attractive.To this end, here an amperometric immunoassay is described for the detection of B. anthracis spores.A moderate power source (5 V via USB connector) supplied a bias voltage (0.4 V) that enabled the measurement of current generated by the reaction between glucose oxidase (GOX, conjugated to anti-B.anthracis spore antibodies) and glucose.2, 6-dichlorophenolindophenol (DCPIP) mediated the electron transfer from GOX to a gold electrode.Magnetic bead-mediated spore capture eliminates the need for a centrifuge and long binding durations.Once more, magnetic bead-conjugated capture antibodies and GOX-conjugated detection antibodies are added in a single step, and substrate incubation occurs at room temperature, further shortening assay duration.Using this strategy, B. anthracis spores can be detected in about 75 minutes.

Materials and Methods
All reagents, supplies, and equipment were purchased from VWR (Wayne, PA) unless otherwise indicated.

Spore Production.
All Bacillus strains used in this research were purchased from BEI Resources (Manassas, VA) and include B. anthracis Sterne strain (cat# NR-1400), B. thuringiensis (NR-610), B. cereus (NR-608), and B. mycoides (NR-612).Each strain was inoculated into 5 mL LB broth (Difco 244620) for 4-8 hours until cloudy before transferring to 50 mL nutrient broth (Fluka 70122).After two days, B. anthracis cultures were transferred to Leighton-Doi broth and were monitored daily until greater than 95% spores were observed via microscopy (Western Digital PMD-1 USB2 1.09, software v 2.0.0,Westover Scientific, Mill Creek WA).Cultures were then centrifuged (30 minutes, 10,000 ×g, 4 ∘ C).Pellets were rinsed once with cold diH 2 O, centrifuged, and then resuspended in 10 mL diH 2 O.One mL aliquots were heat treated before spore concentration was determined via plate counting.Immunoreactivity was confirmed by dot blot analysis (data not shown) though spot intensity did not necessarily correlate with amperometry, immunostrip, or ELISA sensitivity.
Pellets were assayed for GOX activity by incubating in substrate buffer (50 L of 40 M DCPIP, 60 mM glucose in 1.4 M MES, pH 6.2) by inversion for 30 minutes at room temperature and then analyzed on the PHAD instrument.The PHAD software running time was optimized to coincide with the 30-minute incubation.The program instructs the PHAD instrument to perform a series of 22 one second measurement (closed circuit) events, where the largest value from zero is reported (out of 100 measurements per second).Each measurement event is paired with an open circuit interval when buffer exchanges can be performed.The first 10 measurements (A1-A10, see Figure 1) discharge static from the disposable electrode to stabilize the signal; the next 10 cycles (B and C measurements) are used to adjust the baseline signal to maximize the reportable range.The sample is added during the 21st cycle.From this signal the background noise is subtracted.

Instrumentation.
The electrodes were made of 50 nm pure gold sputtered onto 10 mm Toray Lumirror S10 polyester (Materion Corp, Mayfield Heights, OH) backboard.The solder mask (FOC-800 USA, Taiyo America Inc., Carson City, NV) application, laser ablation, and sheet cutting were performed by Conductive Technologies Inc. (York, Pennsylvania).A Plexiglas well (Proto Labs Inc, Maple Plain, MN) is attached via double sided adhesive to form the electrochemical cell.
The PHAD detection box consists of an aluminum housing (2.75  W × 5.5  H × 7.75  D) encompassing a DT9812-2.5Vdata acquisition board with a 4-way multiplexer, 2 kHz oscillator, and LTC6078 dual op-amp (operation amplifier).A 0.4 V bias voltage is applied to one side of the electrochemical cell, while the other side is connected to a current-to-voltage converter through a 10 k Ohm resistor.Thus, the output ampere signal, amplified 10 V/A, is reported as volts.The reportable range of the PHAD box is 250 nA, and resolution is 0.25 nA.
The PHAD box is connected to a PC workstation via USB, which supplies the box with the operational power of 5 V while also performing communication operations.The Kit Instrument software (v0.90) was developed using dotNet

Results
For each run, the PHAD instrument performs several measurements, labeled A, B and C (closed circuit events, Figure 1), which are separated by open circuit intervals when buffers can be exchanged and the offset adjusted.No offset adjustments occur prior to B9 and B10, as these are averaged for the true background signal of the discharged and conditioned electrode.Then, this value is subtracted from the sample signal at B11. Amperometric detection of B. anthracis spores was compared to Tetracore Inc. 's ELISA assay in terms of specificity, limit of detection (LoD), and dynamic range (DR) (Figure 2).Neither PHAD nor ELISA falsely detected 10 7 cfu/mL of B. thuringiensis as B. anthracis.Signals generated for this negative control were similar to signals generated by 10 3 cfu/mL B. anthracis spores; hence, this concentration is below the level of detection of both assays.The LoD for both PHAD and ELISA was 10 4 cfu/mL.The dynamic range ( 2 = 0.975 and 0.995 for PHAD and ELISA, resp.) of both assays was similar (10 3 -5 × 10 4 for PHAD and 10 4 -10 5 cfu/mL for ELISA), though PHAD produced saturated signals at 5 × 10 4 cfu/mL or greater concentrations.ELISA generated saturated signals at spore concentrations of 10 6 cfu/mL and greater (data not shown).In general, PHAD had greater variability in signal output for each spore concentration tested except 10 5 cfu/mL.At this concentration, most signals on the PHAD device are saturated; thus, the true variability for this concentration is not known.LFDs are rapid, disposable chromatographic strips designed for remote testing and therefore were compared to amperometry and ELISA for sensitivity and reliability (Figure 3).LFDs provide no quantifiable data, as any appearance of a line (color development) within the test area of the strips positively indicates the presence of anthrax spores, and color intensity does not necessarily correlate with antigen concentration.Despite differences of sample application (see Section 2), all 3 LFDs (white, light and medium gray bars) performed similarly.All positively detected B. anthracis spores in solutions of 5 × 10 6 cfu/mL for at least 90% of trials.However, the detection rate dropped greatly for lower concentrations (<80% for 10 6 and <60% for 5 × 10 5 cfu/mL).Only New Horizon test strips successfully detected spores at 10 5 cfu/mL or lower, albeit at very low rates (<40% of trials).The subjective interpretation of results is often an understated limitation of LFDs.Tetracore LFDs are the easiest to interpret (usually the test line is dark and highly contrasted), whereas Advnt LFDs almost always produce a very faint test line even at very high spore concentrations (data not shown).ELISA (black bars) had 100% positive detection (zero type 2 errors) for all 25 trials of each spore concentration tested.Amperometry detected spores greater than 90% of trials at concentrations of 10 5 cfu/mL or greater.The rate drops to 86% and 47% for 5 × 10 4 and 10 4 cfu/mL, respectively.
The amount of time to perform an assay is an important parameter in field-detection research.LFDs are unrivaled in time-to-results, with each generating results in as little as 15 minutes of sample application (Table 1).The Tetracore ELISA assay requires over 6 hours for results.The amperometrybased detection described here can obtain results in about 1.5 hours.The LoD of ELISA and PHAD is the same (10 4 cfu/mL) and 100-fold more sensitive than the LFDs.The LoQ, a stricter measure of sensitivity than LoD, is limited to 5 × 10 4 cfu/mL, 5-fold greater than ELISA.

Discussion
Anthrax spores are naturally poorly immunogenic [11].Thus, many commercially available antibodies claiming to be B. anthrax spore specific actually bind to a spore preparation contaminant that originates from vegetative cells [12][13][14].A recent mAb development strategy has attempted to circumvent this problem by developing antibodies that recognize B. anthracis specific sugar moieties [15].However, this strategy presumably only worked with inactivated spores.Spore inactivation can increase or decrease sensitivity of mAb and DNA-based detection assays depending on the method [16].Considering the homogeneity of the Bacillus genera, it is also not surprising that cross-reactivity is a major issue [12,17,18].In our testing, we used a single polyclonal antibody for capture (conjugated to magnetic beads) and 4 monoclonal antibodies for detection (conjugated to GOX).This enabled efficient capture of anthrax spores without falsely detecting other Bacillus species.None of the monoclonals cross-react with the other Bacillus sp.tested (B.cereus, mycoides, and thuringiensis; data not shown); however, specific strains shown to cross-react with monoclonals were not tested [13,15].In addition, fully active spores were used to limit the effects of pretreatment.
ELISAs are a robust, adaptable, and sensitive detection method.Long sample-to-signal times and cost of plate readers have been the ELISA biggest limitation.As the detection assay exits the laboratory and enters the field, the power requirements and delicate optics further limit the traditional and even some more advanced [19] ELISA formats.Amperometry benefits from low cost and power requirements.The PHAD instrument can be made for under $1000 and is powered by laptop battery through a USB cable.For small batch production (less than 10,000 units), the PHAD electrode costs less than $3 and can be reused 2-3 times with minimal drift (data not shown).Here, amperometry was linked with the robustness of antibody-based detection and magnetic-bead capture.Magnetic-bead capture eliminates the need for centrifugation, an activity that is not readily mobilized.The assay was also optimized so that conjugated capture and detection antibodies could all be incubated simultaneously, eliminating several incubation and wash steps typical of ELISA.The reporter enzyme, glucose oxidase, was chosen due to general robustness, a testament to its use in diverse biosensor applications [20,21].Immunoassay robustness is partially attributable to the removal of potential signal interferents prior to substrate incubation via rigorous washing steps.Similarly, the PHAD assay tolerates moderate interferent concentrations (data not shown).The greatest limitation of the current method is the narrow reportable range of the PHAD device.The limit of detection was 1 × 10 4 cfu/mL whereas a maximum signal was achieved with just 5 × 10 4 -1 × 10 5 cfu/mL, just a 5-fold range.The second generation PHAD will have a maximum reportable range of 32 V with a resolution of 1 part in 32,000, thereby enhancing the reportable range and negating the need for background adjustments.

Conclusion
Here, the benefits of generating an amperometric versus colorimetric signal in immunoassay performance were demonstrated.Immunoassay amperometry was comparable to ELISA in terms of limit of detection but was much better in sample-to-signal time.In addition, the low power needs of amperometry allow the assay to be portable, further increasing the appeal of the strategy.The use of magneticbead separation was included to eliminate the need of a centrifuge and thus all high-power equipment.

Figure 1 :
Figure 1: Amperogram of a typical PHAD run.Closed circuit measurements are indicated by a diamond symbol.Otherwise, the PHAD instrument is in "open circuit" mode during which no measurements occur.A1-A10 are used to discharge the electrode.During measurements B1-C10 used blank substrate buffer is replaced with fresh (after C10 blank buffer is replaced with sample substrate buffer) 45-50 seconds prior to subsequent "B" measurement.Between B1 and C8, an offset knob is adjusted after each B, but before subsequent C measurement, to bring background near zero.Averaged background is subtracted from sample signal.

The 10 AFigure 2 :
Figure 2: Linear range of PHAD compared to ELISA.PHAD has a dynamic range between 10 3 and 5 × 10 4 cfu/mL, whereas ELISA has a dynamic range between 10 4 and 10 5 cfu/mL.Spore concentrations that produce statistically saturated signals for PHAD (5 × 10 4 ) and ELISA (10 6 ) are indicated.Average signal for Bacillus thuringiensis (Bt, tested at 10 7 cfu/mL) is also shown adjacent to 10 3 values for comparison.Error bars = standard deviation, dotted line = linear range.
* Assay time does not include initial overnight ELISA-plate coating step.NA: not applicable.