Anal. Chem. 2005, 77, 2882-2888
Comparison of Glycosphingolipids and Antibodies as Receptor Molecules for Ricin Detection Rory Stine and Michael V. Pishko*
Departments of Chemical Engineering, Chemistry, and Materials Science and Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 Cara-Lynne Schengrund
Department of Biochemistry and Molecular Biology, Milton Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033
Glycosphingolipids (GSLs) have been shown to undergo strong interactions with a number of protein toxins, including potential bioterrorism agents such as ricin and botulinum neurotoxin. Characterization of this interaction in recent years has led to a number of studies where GSLs were used as the recognition molecules for biosensing applications. Here, we offer a comparison of quartz crystal microbalance (QCM) sensors for the detection of ricin using antibodies and the GSLs GM1 and asialoGM1, which have been shown to undergo strong interactions with ricin. The presence, orientation, and activity of the GSL and antibody films were confirmed using ellipsometry, Fourier transform infrared spectroscopy (FT-IR), and QCM. It was found that the GSLs offered more sensitive detection limits when directly compared with antibodies. Both GSLs had lower detection limits at 5 µg/mL, approximately 5 times lower than were found for antibodies (25 µg/mL), and their linear detection range extended to the highest concentrations tested (100 µg/mL), almost an order of magnitude beyond the saturation point for the antibody sensors. Potential sites for nonspecific adsorption were blocked using serum albumin without sacrificing toxin specificity. Recent biological attacks involving the toxin ricin1,2 have highlighted the need for a fast, reliable method for detecting potential bioterrorism agents. Typical tests for these toxins involve traditional biochemical methods, such as enzyme-linked immunosorbant assays (ELISA), and require a detection molecule that offers specific recognition of the toxin of interest. Automated detection methods have also been developed for the continuous monitoring of high-risk areas such as airports, post offices, and government buildings.3 Recognition molecules for these devices must not only be sensitive and specific but also robust and capable of monitoring complex environmental samples over long periods of time without significant loss of activity. * To whom correspondence should be addressed. Phone: (814) 863-4810. Fax: (814) 865-7846. E-mail:
[email protected]. (1) Fox, B. The Washington Post, July 29, 2004, p A06. (2) Eggen, D. The Washington Post, February 4, 2004, p A07. (3) Zimmerman, M. The Washington Post, May 26, 2003, p A27.
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Traditionally, antibodies have been used as the detection molecules for these types of biosensors. However, recent studies have shown that glycosphingolipids (GSLs) may be used for sensitive and reliable recognition of certain protein toxins, including ricin, botulinum, tetanus toxin, and cholera toxin.4-13 GSLs offer several potential advantages over antibodies, including increased stability under ambient conditions, ease of orientation at the sensor surface through hydrophobic/hydrophilic interactions, and a larger number of binding sites per unit area compared to the larger antibody. A number of antibody-based techniques for the detection of ricin have been investigated.14-18 Conversely, the use of GSLs in the fabrication of ricin sensors has received only limited attention. While the ricin/GSL interaction has been noted elsewhere,19,20 the only broad work on the subject of GSL-based detection, to our knowledge, was preformed by Gustafson,21 in which the interaction of ricin with a number of GSLs was investigated using surface plasmon resonance. This work qualitatively found that ricin strongly bound to the carbohydrate headgroup of the GSLs GM1 (4) Lauer, S.; Goldstein, B.; Nolan, R. L.; Nolan, J. P. Biochemistry 2002, 41, 1742-1751. (5) Puu, G. Anal. Chem. 2001, 73, 72-79. (6) Singh, A. K.; Harrison, S. H.; Schoeniger, J. S. Anal. Chem. 2000, 72, 60196024. (7) Song, X.; Shi, J.; Swanson, B. Anal. Biochem. 2000, 284, 35-41. (8) Chang, H.; Yang, C.; Yeh, T. Anal. Chim. Acta 1997, 340, 49-54. (9) Fang, Y.; Frutos, A. G.; Lahiri, J. Langmuir 2003, 19, 1500-1505. (10) Song, X.; Swanson, B. I. Anal. Chem. 1999, 71, 2097-2107. (11) Ahn-Yoon, S.; DeCory, T. R.; Baeumner, A. J.; Durst, R. A. Anal. Chem. 2003, 75, 2256-2261. (12) Fisher, M. I.; Tjarnhage, T. Biosens. Bioelectron. 2000, 15, 463-471. (13) Kuziemko, G. K.; Stroh, M.; Stevens, R. C. Biochemistry 1996, 35, 63756384. (14) Rowe-Taitt, C. A.; Anderson, G. P.; Lingerfelt, B. M.; Feldstein, M. J.; Ligler, F. S. Anal. Chem. 2002, 74, 6114-6120. (15) Rowe-Taitt, C. A.; Golden, J. P.; Feldstein, M. J.; Cras, J. J.; Hoffman, K. E.; Ligler, F. S. Biosens. Bioelectron. 2000, 14, 785-794. (16) Narang, U.; Anderson, G. P.; Ligler, F. S.; Burans, J. Biosens. Bioelectron. 1997, 12, 937-945. (17) Rowe-Taitt, C. A.; Hazzard, J. W.; Hoffman, K. E.; Cras, J. J.; Golden, J. P.; Ligler, F. S. Biosens. Bioelectron. 2000, 15, 579-589. (18) Wadkins, R. M.; Golden, J. P.; Pritsiolas, L. M.; Ligler, F. S. Biosens. Bioelectron. 1998, 13, 407-415. (19) Hartley, M. R.; Lord, J. M. Biochim. Biophys. Acta 2004, 1701, 1-14. (20) Sandvig, K.; van Deurs, B. FEBS Lett. 2002, 529, 49-53. (21) Gustafson, I. Colloids Surf., B 2003, 30, 13-24. 10.1021/ac048126s CCC: $30.25
© 2005 American Chemical Society Published on Web 03/18/2005
Figure 1. Chemical structures of GM1 (A) and asialoGM1 (B).
and asialoGM1 (Figure 1), with the stronger binding constant (KD) going to asialoGM1 by approximately 1 order of magnitude. This work, however, did not address the critical issue of detection limit for a sensing element, nor was data presented regarding specificity of the response for the GSL films in the presence of other proteins. Considering the larger number of antibody-based studies that have been preformed, a direct comparison between GSLs and antibodies using identical experimental methods may also prove useful. Here, we present data offering a comprehensive comparison of quartz crystal microbalance (QCM) based ricin sensors utilizing GM1, asialoGM1, and anti-ricin antibodies. The presence and binding capability of the recognition molecules were investigated through surface characterization via ellipsometry and FT-IR, and the issues of detection limit and specificity were explicitly addressed. EXPERIMENTAL SECTION Materials and Equipment. Gold-coated, AT-cut QCM crystals with a base resonant frequency of 10 MHz and an electrode area of 0.2 cm2 were purchased from ICM Co Inc., Oklahoma City, OK. Silicon wafers were purchased from Wafer World, West Palm Beach, FL. Gold coating of the silicon wafers was done by Lance
Goddard Associates, Foster City, CA. The silicon wafers were sputter coated on one side, first with 200 Å of chromium to aid the adhesion of the gold layer to the silicon and then with 1000 Å of gold. Reagent-grade ethanol, concentrated sulfuric acid, hexane, and 30% hydrogen peroxide were purchased from VWR International. Hexadecanethiol (HDT), octanethiol (OT), bovine serum albumin (BSA), fibronectin, bovine IgG, ethyl(dimethylaminopropyl)carbodiimide (EDC), 2-morpholinoethanesulfonic acid (MES), and 11-mercaptoundecanoic acid (MUA) were purchased from Sigma-Aldrich. Ricin B-chain (RTB) and anti-ricin antibody were purchased from Vector Labs, Burlingame, CA. GM1 and asialoGM1 were purchased from Matreya Inc., Pleasant Gap, PA. Ellipsometry measurements were done using a Gaertner LSE ellipsometer at a 70° angle of incidence, utilizing a single wavelength light source from a helium-neon laser. Infrared spectroscopy measurements were done using a Thermo-Nicolet Nexus 670 FT-IR in external reflectance mode with an MCT/A detector. QCM measurements were taken using an ICM 10 MHz lever oscillator and an Agilent digital multimeter and frequency counter. The flow cell used for QCM trials was also purchased from ICM. Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
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GSL Film Deposition. GM1 and asialoGM1 films were first deposited onto gold-coated silicon wafers for ease of surface characterization using ellipsometry and FT-IR. Films were then deposited on gold-coated QCM crystals for affinity sensing experiments. Gold-coated silicon wafers were cleaned by submersion in 3:1 sulfuric acid/hydrogen peroxide solution for 20 min. They were then rinsed sequentially with deionized water and ethanol, sonicated in ethanol for 5 min, rinsed with ethanol again, and dried with a stream of nitrogen. After this, wafers were placed into a solution of 2 mM OT and 2 mM HDT in ethanol and allowed to react for 3 h at room temperature. Brechling and colleagues22 found that a mixed self-assembled monolayer lead to more complete lipid surface coverage with less discontinuities than its homogeneous monolayer counterpart when forming supported lipid films. Due to their protective packaging after production, it was determined that the sulfuric acid/hydrogen peroxide cleaning step could be omitted for the gold-coated QCM crystals. For selfassembled monolayer deposition, the crystals were placed into a specially designed Teflon holder that allowed the gold-coated surface to be exposed while isolating the electrode connections. For the addition of the lipid layer, a procedure described by Meuse and colleagues23 was used, with a few minor modifications. In short, the desired lipid was dissolved in hexane, and this solution was carefully added to a beaker containing water to create a two-phase liquid system. Upon evaporation of the hexane, a lipid film was left at the air/water interface with the hydrophobic fattyacid tail of the lipid oriented toward the air. Finally, the HDT/ OT-coated surface was brought into horizontal contact (parallel to the surface) with the lipid film, creating a single lipid layer on top of the hydrophobic self-assembled monolayer. While no special measures were taken to ensure that a tightly packed lipid monolayer was formed, repeated deposition and subsequent ellipsometry analysis showed the addition of a reproducible lipid film with a thickness ranging from 2 nm for GM1 to 3.2 nm for asialoGM1. These values are comparable to thicknesses reported for lipid monolayer films in the literature.24 Films were then heatstabilized by placement in a desiccated oven at 85 °C overnight. This heat-stabilization step has been found to be critical in reducing shear-induced detachment of the lipid films.25,26 Antibody Immobilization. The cleaning procedure for both gold-coated silicon wafers and gold-coated QCM crystals was identical to the procedure mentioned in the previous section. For antibody immobilization, the gold surfaces were first functionalized by exposure to a 2 mM solution of MUA in ethanol for 3 h at room temperature. The MUA films were then rinsed with ethanol, dried with a stream of nitrogen, and subsequently exposed to a 20 mM solution of EDC in 0.1 M MES buffer (pH 4.5) for 1 h at room temperature. Finally, the activated MUA films were exposed to a 5 µg/mL solution of ricin antibody in 0.1 M PBS (pH 7.2) for 2 h at room temperature, which allowed for the covalent im(22) Brechling, A.; Sundermann, M.; Kleinberg, U.; Heinzmann, U. Thin Solid Films 2003, 433, 281-286. (23) Meuse, C. W.; Krueger, S.; Majkrzak, C. F.; Dura, J. A.; Fu, J.; Connor, J. T.; Plant, A. L. Biophys. J. 1998, 74, 1388-1398. (24) Nyquist, R. M.; Eberhardt, A. S.; Silks, L. A., III; Li, Z.; Yang, X.; Swanson, B. I. Langmuir 2000, 16, 1793-1800. (25) Rowe-Taitt, C. A.; Cras, J. J.; Patterson, C. H.; Golden, J. P.; Ligler, F. S. Anal. Biochem. 2000, 281, 123-133. (26) Stine, R.; Schengrund, C. L.; Pishko, M. V. Langmuir 2004, 20, 65016506.
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mobilization of the antibodies to the surface through amide bonds formed between the terminal carboxylic acid groups of the MUA films and the primary amines found in surface lysine residues on the antibody. Affinity Sensing Experiments. For ellipsometry and FT-IR characterization, a layer of either lipid (GM1 or asialoGM1) or antibody was deposited onto a gold-coated silicon wafer using the above procedure and the surface characterized by ellipsometry and FT-IR. Next, the wafer was placed in a 10 µg/mL solution of RTB in PBS for 2 h at room temperature. After this time, the wafer was removed, rinsed with deionized water to remove nonbound GSL and surface protein, and again characterized by ellipsometry and FT-IR. For QCM experiments, the films were produced as described above and then placed into a 2000 µg/mL solution of BSA in PBS for 3 h at room temperature in order to block any exposed surface sites and prevent nonspecific protein adhesion. The crystal was then placed into the flow cell and allowed to reach a steady baseline frequency. For specificity tests, 100 µL of a 1000 µg/mL IgG solution was first added and allowed to sit at steady state for 5 min. IgG was chosen as a generic protein containing an active sight, thus showing that the film surface was not inherently adhesive to nonspecific proteins. Then, 20 cell volumes of PBS were flushed through the system to remove the IgG solution and any nonbound surface proteins, and the crystal was again allowed to reach a steady frequency. After that, 100 µL of 1000 µg/mL fibronectin solution was added to the cell and the cell was allowed to sit at steady state for 5 min. Fibronectin was chosen as a control protein due to its inherently high adhesion to surfaces, thus making it an excellent test of the specificity of the binding at the sensor surface. Following that, 20 cell volumes of PBS were again flushed through the system and the crystal was allowed to reach a steady frequency. Finally, 100 µL of 1000 µg/mL RTB solution was added to the cell and the cell was allowed to sit at steady state for 5 min. The time frame was chosen to determine the magnitude of the response that could be achieved after a relatively short exposure period and, hence, the sensor’s ability to give a rapid response to the presence of the toxin. After this time, another 20 cell volumes of PBS were flushed through the system, and the crystal was allowed to reach a steady frequency. Frequency response changes were measured as the difference in steady-state frequencies from one rinse step to the next, thus minimizing any viscosity-induced frequency changes caused by the presence of the more viscous protein solutions. For frequency response vs concentration data, 100 µL of the appropriate RTB solution was added to the flow cell and the cell was allowed to sit at steady state for 10 min. Then, 20 cell volumes of PBS were flushed through the system, and the crystal was allowed to reach a steady frequency. Frequency response changes were measured as the difference in steady baseline frequencies from one rinse step to the next. RESULTS AND DISCUSSION Film Characterization. Table 1 shows ellipsometry data that was collected for antibody, GM1, and asialoGM1 films on goldcoated silicon wafers and depicts film thickness increases for each successive layer. For the antibody sample described in Table 1, a distinct increase in film thickness of 2.5 nm was noted upon covalent addition of the antibody to the MUA film. This value
Table 1. Ellipsometry Data Showing the Increases in Film Thickness for the Addition of Each Film Layer for the Antibody, GM-1, and AsialoGM1 Deposition thickness (nm) SAM
antibody GM1 asialoGM1 a
receptor molecule
RTB
total
errora
increase
total
errora
increase
total
errora
increase
1.1 1.5 1.2
(0.1 (0.1 (0.5
1.1 1.5 1.2
3.6 3.5 4.4
(1.2 (0.8 (1.1
2.5 2.0 3.2
5.6 5.2 6.5
(1.7 (1.0 (1.1
2.0 1.7 2.0
Error bars represent standard deviations over eight trials.
agreed within the wide range of values reported for protein film thickness, ranging from 1.5 to 5 nm.27,28 After exposure to a solution of RTB, another distinct 2 nm increase in film thickness was noted, indicating that the antibody remained active and capable of binding its target analyte after the immobilization process. For the GM1 sample in Table 1, the addition of the GM1 layer to the mixed HDT/OT self-assembled monolayer showed an increase of approximately 2 nm, a value that is in agreement with previously reported film thickness measurements for supported lipid monolayers.24 The final sample presented in Table 1 shows that upon addition of the asialoGM1 layer, a thickness increase of 3.2 nm was observed. While this still falls within the bounds of reported values for such films,23 it was a statistically significant variation from the value found for the GM1 film. Referring back to Figure 1, this may be explained by examining the structures of the two GSLs. The removal of the sialic acid side chain in asialoGM1 could allow for tighter packing of the lipid monolayer on the surface. This would lead to a reduction of empty space in the film and, hence, a larger reading via ellipsometry due to the manner in which this technique measures thickness. Ellipsometry calculates film thickness by monitoring the relative change in rotation of two ellipsometrically polarized laser beams as they pass through the film. If empty space (air) is present in the film, this will act to lower the refractive index from the expected value, thus leading to a lower calculated value for total film thickness. This tighter packing also may force the lipid molecule into a more extended conformation, thereby causing an actual physical increase in film thickness. Upon exposure of the GSL films to a solution of RTB, distinct thickness increases of 1.7 and 2 nm, for the GM1 and asialoGM1 samples, respectively, were again noted. This would seem to indicate that the heatstabilizing process did not harm the GSL films and both are properly oriented at the sample surface and capable of being bound by RTB. It is also interesting to note that the thickness increases that were observed for all three samples after their exposure to the RTB solutions are statistically identical within the 95% confidence intervals. This most likely indicates that the films of all three recognition molecules maintained enough active sites per unit area to permit the saturation of the surface with RTB when given 2 h to bind. This finding will be touched upon later when we examine sensor detection limits over a much shorter time. (27) Facci, P.; Andolfi, D. A., L.; Schnyder, B.; Kotz, R. Surf. Sci. 2002, 504, 282-292. (28) Berzina, T. S.; Petrigliano, V. I. T., A.; Alliata, D.; Tronin, A. Yu.; Nicolini, C. Thin Solid Films 1996, 284-285.
Figure 2 presents FT-IR data taken for the same GM1 (a) and asialoGM1 (b) films, both before and after exposure to RTB. The GM1 scan (Figure 2a, top) showed several peaks confirming the presence of the carbohydrate headgroup. The large peak at 1079 cm-1 was indicative of the oxygen-containing rings that make up the bulk of the headgroup. There was also a sharp peak present at 1265 cm-1, corresponding to C-O bonding, and a small peak at 1639 cm-1, indicating the presence of the amide bonds in the GM1 structure. A distinct change can be seen in the scan of the GM1 film after exposure to RTB (Figure 2a, bottom). The sharp peak at 1265 cm-1 had expanded to include a large shoulder region that stretches to 1400 cm-1. This was most likely due to the addition of a large number of C-N bonds from the added protein layer. Most tellingly, however, was the addition of the large amide I and amide II doublet at 1659 and 1546 cm-1, respectively. This is the region that is commonly investigated to determine the presence of a protein with FT-IR, since amide bonds are the predominant feature of polypeptides. Aside from the increased intensity, the position of the amide I peak had also shifted from 1639 cm-1 in the GM1 scan to 1659 in the GM1 + RTB scan. This shift corresponds to the change that would be expected when going from a predominantly alkyl amide, such as those found in GM1, to a predominantly polypeptide amide, such as would be expected for a protein film. The FT-IR scan of the asialoGM1 film (Figure 2-b top) showed some interesting differences from its GM1 counterpart. The large oxygen-containing ring was still present, but the sharp C-O peak at 1265 cm-1 diminished. This would seem to indicate that this peak was the result of the C-O bond in the carboxylic acid group found in the sialic acid residue of GM1. The amide peak at 1639 cm-1 had also disappeared and been replaced by what appears to be a CdO a peak at 1780 cm-1. The significant effect that the removal of the sialic acid residue had on the FT-IR scan may suggest that the GM1 film orients itself in a manner that presents the sialic residue at the surface. This would account not only for the shift in FT-IR peaks, but also the perceived increase in film thickness as measured with ellipsometry. If the carbohydrate headgroup of GM1 contorts itself to offer the sialic acid residue at the interface, it would most likely lead to an increase in surface area per GM1 molecule and therefore a less densely packed film than the asialoGM1 sample. After exposure of the asialoGM1 film to RTB (Figure 2b, bottom), we again saw the tell-tale peaks associated with a protein film, specifically the broad C-N peak stretching from 1265 to 1376 cm-1 and the amide I and amide II peaks at 1642 and 1554 cm-1, respectively. When the FT-IR and Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
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Figure 2. FT-IR/ERS spectra for GM1 (A) and asialoGM1 (B) films both before and after exposure to RTB. The GSL films (top scans in each) show peaks indicative of the presence of the carbohydrate headgroups, particularly the oxygen containing rings shown by the large peaks at 1075 cm-1. After exposure to RTB (bottom scan in each), new peaks are noted that indicate the presence of a protein layer, particularly the amide I and amide II peaks at 1659 and 1546 cm-1, respectively.
ellipsometry data collected for each of these samples was considered, it offered strong evidence of the proper orientation and binding ability of the films, as well as offering some insight into potential structural differences in each sample. Affinity Sensing Experiments. Specificity trials were performed to determine the relative response of the three recognition molecules in the presence of RTB and two controls (IgG and fibronectin). Figure 3 shows the results of these experiments. None of the three recognition molecules gave a significant response to IgG, with the 95% confidence intervals falling within the bounds the instrument background noise. The experiments run utilizing the fibronectin control, however, showed some variation in the frequency changes observed. A significant frequency change was observed for antibody sensors in the presence of fibronectin, close to the magnitude of that obtained 2886 Analytical Chemistry, Vol. 77, No. 9, May 1, 2005
for RTB. An ELISA was preformed (data not shown) and confirmed a substantial degree of cross-reactivity between the antibody and fibronectin. The specificity that was observed for fibronectin was almost half of that observed for RTB. Experiments showed that over the 5-min detection period, this cross-reactivity was enough to give an almost equivalent response between the fibronectin control and the analyte of interest. For the GM1 sensing elements, the response obtained from the fibronectin trials was insignificant from the background noise of the instrument. However, asialoGM1 trials did show a small but statistically significant response. The difference between the responses for the GM1 and asialoGM1 sensors is most likely due to the removal of the charged sialic acid residue in asialoGM1. This negatively charged group would aid in the repulsion of the negatively charged protein, essentially allowing for a total rejection
Figure 3. Specificity trials for each of the three recognition molecules in the presence of RTB, fibronectin, and IgG. The control proteins do not show a significant response with the exception of fibronectin with the anti-ricin antibody, which was confirmed to have cross-reactivity with fibronectin through an ELISA. The relative magnitudes of the sensor responses to RTB show the antibody with the weakest sensitivity, 3 times lower than for GM1 and 10 times lower than for asialoGM1. Error bars represent standard deviations over five trials.
Figure 4. Detection ranges for the three recognition molecules used. The poorest performance was seen in the antibody-based sensors, with a lower detection limit of 25 µg/mL and a loss of linear detection above 50 µg/mL. The GM1 and asialoGM1 sensors gave similar lower limits at around 5 µg/mL and offered linear detection up to the highest concentration tested. Asialo-GM1 gave the strongest response at higher concentrations, but declined more rapidly, leading to similar responses between GM1 and asialoGM1 at lower concentrations.
of fibronectin from the surface. When this group is absent, as in the asialoGM1 samples, the now neutrally charged surface is more susceptible to fibronectin adhesion, hence the small positive response. Responses to the RTB analyte varied widely among the three recognition molecules. The weakest average response was obtained for the anti-ricin antibody, with the magnitude of the frequency response falling approximately 3 times below that observed for GM1 and 10 times below asialoGM1. In the light of the aforementioned ellipsometry data, which showed that all three recognition molecules obtained statistically identical saturation thicknesses after 2 h, this would seem to suggest that the differences observed in the magnitudes of the responses for each sample are the result of the strength of the individual binding interactions and not due to a lack of binding sites. This is a particularly important aspect to consider when developing a sensor for a potential bioterrorism threat, as fast response time is a must to ensure that the hazard is detected in a time frame that will minimize exposure. Experiments were also performed to determine the detection range of the sensing elements. These data are presented in Figure 4. The antibody trials clearly appear to have produced the poorest linear detection range of the three recognition molecules used. The lower detection limit of the sensor was reached at a concentration of approximately 25 µg/mL (0.7 µM) of RTB. Experiments were run at varying concentrations up to 100 µg/ mL; however, the antibody-based sensors began to saturate above RTB concentrations of 50 µg/mL and no longer provided a linear response. The relatively poor lower detection limit came as somewhat of a surprise, as previous studies utilizing antibodybased QCM sensors for other analytes have achieved somewhat better results.29,30 These tests, however, were not run under identical conditions to those used here, and the KD values for the antibodies used in these studies were not given; hence, a completely unbiased comparison cannot be made. One method
that was attempted to increase the range of our antibody sensors was to orient the active sites of the antibodies on the QCM crystal surface using protein G. Experiments (data not shown) indicated that this procedure approximately doubled the RTB response of the antibody sensors to a magnitude that was comparable with that obtained for GM1. Unfortunately, this benefit was greatly outweighed by an almost 10-fold increase in nonspecific binding. When specificity tests were run using both RTB and fibronectin, the large nonspecific response was overwhelming, thereby preventing any conclusions from being made about toxin binding. The data that was obtained for the GM1 experiments indicated a lower detection limit of 5 µg/mL (0.1 µM) and a linear sensing range up to 100 µg/mL. This detection range was in line with previous studies utilizing GM1-based QCM sensors for the detection of cholera toxin.26,31,32 Trials for the asialoGM1 sensors presented an interesting set of results. At the highest concentrations tested, the magnitude of the response from these sensors was more than 4 times higher than for GM1; however, the lower detection limit was found to be approximately at the same concentration that was found for the GM1 sensors. This meant a significantly increased sensitivity (change in signal per change in sample concentration) for the asialoGM1 sensors over their GM1 counterparts, as evidenced by the greater slope for the asilaoGM1 experiments shown in Figure 5. In comparing the results for sensing range from the three recognition molecules tested, it is clear that asialoGM1 offered the most useful linear response, with both the lowest detection limit and largest variation of sensor response for a given change in RTB concentration. This is in agreement with the work of Gustafson,21 who found that asialoGM1 underwent a stronger binding interaction with ricin than did GM1. The comparatively better performance of the two GSL-based sensors over the antibody may be related to the relative size of the recognition molecules. As mentioned in the Introduction, the smaller area occupied by an individual GSL molecule leads to a larger number of toxin binding sites per unit surface
(29) Lu, H. C.; Cen, H. M.; Lin, Y. S.; Lin, J. W. Biotechnol. Prog. 2000, 16, 116-124. (30) Ben-Dov, I.; Willner, I.; Zisman, E. Anal. Chem. 1997, 69, 3506-3512.
(31) Spangler, B. D.; Tyler, B. J. Anal. Chim. Acta 1999, 399, 51-62. (32) Spangler, B. D.; Wilkinson, E. A.; Murphy, J. T.; Tyler, B. J. Anal. Chim. Acta 2001, 444, 149-161.
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area and may explain, at least in part, the stronger signal obtained from the GSL sensors. It is evident that the lower detection limits reached in this work may not be sufficiently sensitive to be utilized in the type of monitoring system necessary to alert the public to dangerous levels of toxin in the event of an attack. The limit that was reached, however, is attributable more to the limitations of the QCM platform than to the limits of the recognition molecules.33,34 This is evident if the well-characterized GM1/cholera toxin interaction is considered. Lower detection limits for GM1-based QCM sensors for cholera toxin, as previously mentioned, are in agreement with the lower detection limits found in this work for ricin (5 µg/mL). If more sensitive signal transduction methods are used, though, a considerably lower limit can be reached. Fluorescent detection methods utilizing the same GM1/cholera toxin interaction have been capable of significantly lower detection limits,7,25 as much as 3 orders of magnitude, and similar transduction methods could most likely be used with the GSL/RTB interaction to obtain more sensitive results. CONCLUSIONS Here, we have presented for the first time a clear comparison of the capabilities of GSL- and antibody-based detection of ricin. (33) O’Sullivan, C. K.; Guilbault, G. G. Biosens. Bioelectron. 1999, 14, 663-670. (34) Davis, K. A.; Leary, T. R. Anal. Chem. 1989, 61, 1227-1230.
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This information may be of particular importance in the development of environmental sensors as an early alert method in the case of a terrorist attack. The recognition molecules were immobilized on the surfaces of gold-coated QCM crystals, and their presence and ability to bind or be bound by the B-chain of ricin was confirmed through surface characterization methods. The GSL sensors appear to have outperformed their antibodybased counterparts, offering lower detection limits and better sensitivity. The lower limit of these sensors, found to be in the 10-6 g/mL range, could most likely be improved through the use of a more sensitive transduction method, such as fluorescence or electrochemical impedance. ACKNOWLEDGMENT This work was supported by the NIH (EB000684-01), the NSF (BES-0426170), and the Commonwealth of Pennsylvania. This project is also funded, in part, under a grant with the Pennsylvania Department of Health’s Health Research Formula Funding Program (State of Pennsylvania, Act 2001-77-PA Tobacco Settlement Legislation). The Department specifically disclaims responsibility for any analyses, interpretations, or conclusions. Received for review December 20, 2004. Accepted February 15, 2005. AC048126S
