Environ. Sci. Technol. 2000, 34, 2845-2850
Automated Fiber Optic Biosensor for Multiplexed Immunoassays KEELEY D. KING,† JESSICA M. VANNIERE,‡ JENNIFER L. LEBLANC,‡ KAREN E. BULLOCK,‡ AND G E O R G E P . A N D E R S O N * ,‡ Geo-Centers, Inc., 1801 Rockville Pike, Suite 405, Rockville, Maryland 20852, and Naval Research Laboratory, Center for Bio/Molecular Science and Engineering, Code 6910, Washington D.C. 20375-5348
The multianalyte capability of the RAPTOR, a rapid, automatic, and portable fiber optic fluorimeter, was demonstrated. Employing evanescent wave illumination on polystyrene fiber optic waveguides, the RAPTOR performed fluorescent immunoassays for Bacillus globigii spores, ovalbumin, Erwinia herbicola, and MS2 coliphage. During a 4-day laboratory trial assaying 144 blind samples, the RAPTOR demonstrated detection of 105 cfu/mL B. globigii, 107 cfu/mL E. herbicola, and 109 pfu/mL MS2; ovalbumin detection was less favorable than expected due to sample degradation. Assays were completed in 10 min with no sample preprocessing. No false positives were identified. Antigen carryover between coupons was examined but was not found to elicit a notable response for any analytes, except B. globigii. Finally, assay results obtained after reagent and waveguides had completed 30 negative (buffer) cycles were compared with standard assay results achieved with fresh reagent and waveguides to determine whether antigen detection would decrease using cycled reagent or optical probes. Detection efficacy proved to be unaffected by the use of cycled versus fresh probes or reagent.
Introduction The potential threat of exposure to toxic or infectious biological material has spurred the development of improved detection systems. Effective defense against biological threats requires rapid detection and identification methods that permit prompt donning of protective clothing and early medical intervention. To this end, the development and production of a forward deployable instrument with identification capabilities for rapid detection of biological threats in clinical and environmental samples is imperative. Antibody-based identification has been exploited in the development of numerous sensor platforms because of its high sensitivity, specificity, and adaptability to field use (1). Piezoelectric crystal sensors that monitor the frequency-tomass ratio of antigen bound to antibody immobilized to the surface of quartz crystals have been used for the detection of ricin (2), Vibrio cholerae (3), and Staphylococcal Enterotoxin B (SEB) (4, 5). SEB and ricin have also been successfully * Corresponding author phone: (202)404-6033; fax: (202)767-9594; e-mail:
[email protected]. † Geo-Centers, Inc. ‡ Naval Research Laboratory. 10.1021/es9913535 CCC: $19.00 Published on Web 06/02/2000
2000 American Chemical Society
assayed using an integrated optic interferometer (6). Additionally, detection of pathogenic agents has been achieved with evanescent wave-based fiber optic biosensors (7-10). Antibodies have even been used in sensors for the detection of explosives such as TNT and RDX (11, 12) or environmental pollutants such as pesticides (13). Ideally, a field-deployable instrument should identify substances with limits of detection less than 0.1 µg/L, provide assay times of less than 1 h, and have the capability for repeated use while being able to recognize a wide range of substances concurrently (1, 14). As successful as the aforementioned instruments were, they either performed singleanalyte detection only or they were manually operated. More recently, an antibody-based array biosensor for the simultaneous identification of SEB, F1 antigen from Yersinia pestis, and the sepsis marker D-Dimer was described (15). Using antibodies arranged in discrete patterns on silica microscope slides, this system has promising multianalyte capabilities but has not yet been automated for field applications. At the same time a precursor to the system described here, the Analyte 2000 fiber optic biosensor (Research International, Woodinville, WA), was automated to perform remote detection of bacterial spores on an unmanned airplane (16, 17). However, it was not engineered for routine field-testing. In an effort to produce a portable, compact, and automated instrument with multianalyte capabilities, a fiber optic biosensor prototype, the MANTIS, was introduced (18). After numerous improvements, the MANTIS was eventually renamed RAPTOR (Figure 1). The third device in the lineage of such biosensors, the RAPTOR, is a compact (18.6 cm × 27.4 cm × 17.3 cm), lightweight (5.45 kg/12 lbs), selfcontained, multichannel instrument capable of performing four sandwich fluoroimmunoassays simultaneously on the surface of 4 cm polystyrene fiber optic waveguides (19). Few biosensor systems have demonstrated the capability for parallel testing of different classes of analytes. An array biosensor for the simultaneous identification of bacterial, viral, and protein analytes has recently been described (20). Detection of Erwinia herbicola vegetative cells, Bacillus subtilis spores, and male-specific coliphage (MS2) has also been achieved simultaneously using a microchip PCR array instrument (21). Previously, the RAPTOR exhibited its ability to discriminate between bacterial and protein analytes when it participated in a blind study interrogating samples for SEB, ricin, Bacillus globigii, and Francisella tularensis (19). At this time we are presenting the results of the RAPTOR’s performance in a blind trial which evaluated several biosensors’ capabilities to detect two bacteria, Bacillus globigii (Bg) and Erwinia herbicola (Eh), as well as ovalbumin and MS2 virions in buffer. Though ubiquitous in nature and relatively innocuous in themselves, these analytes serve as simulants of serious pathogens and toxins, such as Bacillus anthracis, Yersinia pestis, SEB, ricin, and Venezuelan equine encephalitis (21). Following the lab trial, additional experiments were conducted with the RAPTOR to assess the possibility of antigen carryover between consecutive assays. Then, to investigate continuous monitoring capabilities, the effect of repeated assay cycles on reagent and coupon sensitivity were studied.
Experimental Methods and Procedures Buffers and Reagents. Antibody and antigen stocks were supplied by the Naval Medical Research Center (NMRC) in Bethesda, MD. RAPTOR wash buffer (PBT) consisted of phosphate buffer (Sigma, 8.3 mM, pH 7.3) containing 0.05% VOL. 34, NO. 13, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. The RAPTOR. Disposable coupons into which antibodycoated optical probes are secured are inserted into the center hatch. The right-most compartment houses on-board reservoirs for buffer, waste, and fluorescent reagent. Initiation of assay protocols, or “recipes,” is accomplished through the keypad at the left. At the conclusion of an assay, results are displayed in the LCD panel. In the field the portable biosensor can be operated for over 8 h using its own BA-5590/U battery. (v/v) Triton X-100 and 0.01% (w/v) sodium azide (NaN3). All solutions were prepared using deionized water and reagent grade chemicals. Additional reagents such as casein, bovine serum albumin (BSA), and Triton X-100 (TX-100) were also purchased from Sigma (St. Louis, MO). Probe Preparation. Immobilization of primary antibody to fiber optic waveguides (Research International) was achieved by passive adsorption of 100 µg/mL IgG in sodium carbonate buffer (0.1 M Na2CO3, pH 9.6) overnight at 4 °C. Prior to antibody functionalization, however, the distal tips of the probes were blackened to prevent reflection of excitation light. Capillary tubes (100 µL) cut into 4-cm pieces were filled with 36 µL of the appropriate antibody solution. After capping one of the exposed ends (Supelco column chromatography cap, 1/16′′ i.d.), probes were carefully inserted and refrigerated overnight to allow for adsorption. Coupon Assembly. After overnight incubation, unbound IgG was rinsed off by briefly immersing the probes in dH2O. The probes were then ready to be mounted into injectionmolded, disposable assay cartridges called “coupons”. Described in detail previously (19), the probes are inserted into the coupon and glued into place using UV-curing epoxy (Norland Optical Adhesive 68). Each coupon was then coded by coloring a series of squares on the back of the coupon with white and black paint pens corresponding to a particular assay recipe that was to be run on the RAPTOR. Preparation of Cy5-Labeled Antibodies. Cy5-conjugated antibodies were prepared by reacting 3 mg of protein (1 mg/ mL) in 0.05 M sodium tetraborate, 0.04 M NaCl, pH 9.0 with one vial bisfunctional Cy5-reactive dye (λex ) 649 nm, λem ) 670 nm; Amersham Life Science Products, Arlington Heights, IL) for 30 min at room temperature in the dark. Subsequently, labeled protein was separated from unincorporated dye by size-exclusion chromatography using a BioGel P-10 column (Bio-Rad, Hercules, CA) equilibrated with PBS, pH 7.4. Final protein concentration and dye-to-protein (D/P) molar ratios were calculated based on the A280 and A650 as described by the manufacturer. Ideal D/P ratios ranged from 2 to 6 Cy5 molecules per IgG. Prior to an assay the Cy5-reagent was diluted in blocking buffer containing 1 mg/mL casein and 1 mg/mL BSA in PBS (pH 7.4) with 0.1% (v/v) TX-100 detergent added. RAPTOR Design and Operation. Inserting a coupon into the RAPTOR aligns all necessary optical paths and engages 2846
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the required fluidic connections via blunt tipped needles in the RAPTOR’s coupon compartment that penetrate a rubber septum on the underside of the assay coupon. A pneumatic pump moves buffer, air, fluorescent reagent (from on-board reservoirs), or sample within the system. Serpentine channels in the coupon provide a common path across the probe surfaces. Bubble detectors, which monitor liquid-to-air interfaces, control introduction of sample and fluorescent reagent. During the two-step sandwich immunoassay, antigen present in a sample binds to fiber optic probes coated with capture antibody specific for that antigen; any unbound material is washed away following a brief rinse with PBT. Typically, 1 mL of an aqueous sample is injected manually into a sample port (3 mL capacity); however, samples can also be introduced automatically from a cyclone aerosol collector controlled by the RAPTOR. Fluorescently labeled (Cy5) antibody (the “reagent”), which is maintained at a suitable temperature in a removable thermal storage module and recovered after each assay cycle, is introduced and binds to the antibody-antigen complexes on the probe surface, completing the sandwich assay. Excitation light from four 5 mW Sanyo laser diodes (635 nm) within the RAPTOR is focused into the custom injection-molded waveguides. An evanescent wave is created along each probe, exciting the fluorescent emission of specifically bound Cy5-labeled antibodies. The portion of the fluorescence captured by the optical probe is collimated by the probe’s molded lens and focused onto a photodiode using a ball lens, chosen for its light-gathering power and short focal length. A long-pass dichroic filter (665 nm) is incorporated to reject reflected laser light. In this way, one sample is interrogated for four different analytes simultaneously. All instructions regarding fluidics, data collection, and data analysis parameters are defined by specially written “recipes” loaded into the RAPTOR, which has a 386 microprocessor with 1 Mb of memory for data storage. In addition, each coupon’s identity regarding assay protocols to be utilized is defined by a bar code made up of an array of black and white squares, which is read upon insertion of the coupon into the RAPTOR. Up to 63 different coupon designations can be entered for use by the instrument. Assays are initiated by pressing the “Run Assay” command on the RAPTOR’s keypad. The results of each assay, which range in duration from a rapid, 3-min screening assay to a 10-min standard assay, are ultimately reported as negative (NEG), suspect (SUS), positive (POS), or high positive (HI+), along with the identity of the agent, in a liquid crystal display window on the face of the RAPTOR. Two parameters are considered when determining assay results: “assay rate” and “wash delta”. The assay rate refers to the slope of the fluorescent signal increase during a 90 s binding period of the labeled antibody, while the wash delta represents the signal increase in picoAmps (pA) over baseline due to bound fluorescent reagent after each probe has been washed free of excess Cy5-labeled antibody. While one parameter is sufficient for strongly positive samples, use of both factors greatly reduces the number of false positives obtained for determinations near the limit of detection. All assay data are stored by the RAPTOR for later review. Assay Procedure. At the beginning of a standard assay, prior to analyte challenge, the RAPTOR automatically initiated a 5-min baseline recipe to establish a background level representing the nonspecific adsorption of the Cy5-reagent to the probes. An initial background rate was determined during a 90 s fluorescent antibody incubation period. After reagent recovery, the probes were rinsed with PBT, and an initial background wash value was determined. Once background levels were obtained, a blank sample (dH2O) was injected.
TABLE 1. Sandwich Immunoassay Components and Cutoff Values SUS
POS
HI+
analyte
capture antibody 100 µg/mL
detection (Cy5) antibody 10 µg/mL
assay rate
wash delta (pA)
assay rate
wash delta (pA)
assay rate
wash delta (pA)
B. globigii (Bg) ovalbumin (Ova) E. herbicola (Eh) MS2
APa goat anti-Bg IgG AP rabbit anti-Ova IgG AP goat anti-Eh IgG rabbit anti-MS2 IgG
Cy5 rabbit anti-Bg IgG Cy5 AP rabbit anti-Ova IgG Cy5 AP goat anti-Eh IgG Cy5 rabbit anti-MS2 IgG
20 20 20 15
40 30 40 40
40 30 40 20
80 60 80 60
100 100 100 100
200 200 200 200
a
Affinity purified.
Samples were loaded into the sample port using a 1 mL syringe affixed with a blunt-tipped needle. An assay consists of numerous steps, all of which were performed automatically by the RAPTOR. First the coupon was rinsed briefly with PBT wash buffer. Then antigen was allowed to bind to the optical probes during a 7-min sample incubation period. Afterward, unbound material was eliminated by rinsing the coupon with PBT. At the same time, the sample port was also flushed out. Next the coupon was cleared with air to prevent dilution of the incoming reagent. Then Cy5-antibody reagent was loaded for 90 s to interrogate the amount of antigen bound to the probes. The rate of fluorescence increase during incubation with the Cy5-antibody was calculated, and the reagent was returned to its reservoir for reuse. The coupon was flushed with wash buffer one last time, and a final reading was taken to determine the increase in fluorescence due to Cy5-antibody bound during the assay cycle. Including all wash steps, an entire standard assay cycle was completed in 10 min. Single-Analyte Assays. Single analyte assays were conducted prior to the trial to select the best antibody combinations for each assay. Each antibody available was tested as both a capture antibody and as the Cy5-antibody reagent. Coupons consisting of one control probe and three test probes coated with antibody against a single analyte were prepared. The fluorescent reagent contained 10 µg/mL of Cy5-labeled antibody specific for one analyte only. Dose responses of the three antigen concentrations expected to be examined during the trial were tested during the single analyte assays: Bg 104, 105, and 106 cfu/mL; ovalbumin 2, 20, and 200 ng/mL; Eh 107, 108, and 109 cfu/mL; and crude MS2 107, 108, and 109 pfu/mL. All antigen solutions were prepared in PBS with 0.01% (v/v) TX-100. Multianalyte Assays. Based on data gathered from the single analyte assays, multianalyte assays were conducted whereby each optical probe within the coupon was specific for one of the four analytes to be assayed. Table 1 lists the antibody combinations selected for the four sandwich immunoassays. Detection was accomplished using a cocktail of 10 µg/mL of each of four Cy5-labeled antibodies. Although experiments were performed comparing 5 µg/mL to 10 µg/ mL of each Cy5-antibody in the reagent cocktail, data collected during the multianalyte tests verified that 10 µg/ mL of each Cy5-IgG provided adequate detection without serious crossreactivity issues. The use of an antibody cocktail as the Cy5-antibody reagent was similarly validated by Rowe et al. (15). In this way, one sample could be tested for four separate analytes simultaneously within a single coupon. Only the two less concentrated dilutions of each simulant were investigated here: Bg at 104 and 105 cfu/mL; ovalbumin at 2 and 20 ng/mL; Eh at 107 and 108 cfu/mL; and crude MS2 at 107 and 108 pfu/mL. Decisions concerning limits of detection, wash delta, and assay rate cutoffs were also made based on the results of the multianalyte assays; assay rate and wash delta parameters were determined empirically and can be modified to suit the sensitivity of any assay (Table 1). Blind Trial. The blind trial involved the interrogation of 144 unknown samples for the presence of Bg, ovalbumin,
Eh, or MS2. Antigen samples received from NMRC were kept frozen prior to testing; fresh Cy5 reagent cocktail was prepared at the Naval Research Laboratory daily. Two RAPTOR units were employed to test 36 samples per day over a 4-day period. Assay coupons assembled in preparation for the trial were kept refrigerated throughout the duration of the testing. At the beginning of each day of testing, 36 new samples were removed from the freezer and allowed to thaw at room temperature for 20-30 min. Prior to being assayed, each sample was sonicated for 1-2 min. Coupons were used repeatedly until a sample was identified as POS or HI+ for one of the analytes; suspect identifications were treated as negative responses and the trial continued uninterrupted. After positive identification, the system was flushed two times by manually selecting a “Flush system” command on the RAPTOR’s keypad, even though the RAPTOR automatically purges the system at the conclusion of each assay cycle. Then the coupon was removed and replaced with a fresh one; any time a coupon was replaced the RAPTOR automatically reset the baseline for the new coupon. Results for each sample were recorded and submitted to a trial monitor daily. Blind samples to be interrogated included the following concentrations: Bg at 104, 105, and 106 cfu/mL; ovalbumin at 2, 20, and 200 ng/mL; Eh at 107, 108, and 109 cfu/mL; and MS2 at 107, 108, and 109 pfu/mL. Ten replicates of each concentration and 24 blank samples constituted the blind sample set. Sample Carryover and Cross-Contamination. Antigen carryover from coupon to coupon was evaluated with used coupons saved from the blind trial. A coupon could be used to retest any analyte except the one for which it was already positive. Each simulant was studied one at a time, and each experiment involved three coupons run sequentially. Upon insertion of the first coupon, a high concentration of antigen was injected, and a standard response was generated. Without performing an additional system flush, a second coupon was inserted, and the assay was repeated using buffer as the sample. Afterward, the system was flushed once, another 1 mL of buffer was injected, and a third assay was initiated. Analyte concentrations investigated were as follows: 108 cfu/ mL Bg, 5 ug/mL ovalbumin, 109 cfu/mL Eh, and 1010 pfu/mL MS2. Negative Buffer Cycling. To mimic field conditions in which the RAPTOR would be performing for extended periods of time, experiments were conducted whereby a 10-min assay cycle was automatically repeated 30 consecutive times. At the conclusion of the 30 negative cycles, 106 cfu/mL Bg, 200 ng/mL ovalbumin, 107 cfu/mL Eh, and 109 pfu/mL MS2 were all assayed using the coupon and the reagent cocktail that had endured the repetitive cycles. Then, the coupon was replaced with a fresh one and the same antigen samples were again assayed still using the same reagent which, by this time, had been through approximately 35 cycles. Ultimately the results of these two experiments were compared to standard responses achieved when each antigen sample was interrogated using fresh reagent and fresh coupons in order to determine whether sensitivity was VOL. 34, NO. 13, 2000 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Trial Results analyte Ova MS2 Eh Bg blank
concentration
no. of positives (predicted)
no. of positives (actual) n ) 10
% correctly identified
2 ng/mL 20 ng/mL 200 ng/mL 107 pfu/mL 108 pfu/mL 109 pfu/mL 107 cfu/mL 108 cfu/mL 109 cfu/mL 104 cfu/mL 105 cfu/mL 106 cfu/mL 0
0 10 10 0
