Rapid Simultaneous Ultrasensitive Immunodetection of Five Bacterial

Article pubs.acs.org/ac

Rapid Simultaneous Ultrasensitive Immunodetection of Five Bacterial Toxins Yuri M. Shlyapnikov,*,† Elena A. Shlyapnikova,† Maria A. Simonova,‡ Anna O. Shepelyakovskaya,§ Fedor A. Brovko,§ Ravilya L. Komaleva,‡ Eugene V. Grishin,‡ and Victor N. Morozov† †

Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region, Russia 142290 Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia 117997 § Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, Russia 142290 ‡

S Supporting Information *

ABSTRACT: Rapid ultrasensitive detection of gastrointestinal pathogens presents a great interest for medical diagnostics and epidemiologic services. Though conventional immunochemical and polymerase chain reaction (PCR)-based methods are sensitive enough for many applications, they usually require several hours for assay, whereas as sensitive but more rapid methods are needed in many practical cases. Here, we report a new microarray-based analytical technique for simultaneous detection of five bacterial toxins: the cholera toxin, the E. coli heat-labile toxin, and three S. aureus toxins (the enterotoxins A and B and the toxic shock syndrome toxin). The assay involves three major steps: electrophoretic collection of toxins on an antibody microarray, labeling of captured antigens with secondary biotinylated antibodies, and detection of biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shearflow. All the stages are performed in a single flow cell allowing application of electric and magnetic fields as well as optical detection of microarray-bound beads. Replacement of diffusion with a forced transport at all the recognition steps allows one to dramatically decrease both the limit of detection (LOD) and the assay time. We demonstrate here that application of this “active” assay technique to the detection of bacterial toxins in water samples from natural sources and in food samples (milk and meat extracts) allowed one to perform the assay in less than 10 min and to decrease the LOD to 0.1−1 pg/mL for water and to 1 pg/ mL for food samples.

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not only as bacterial biomarkers: being extremely hazardous, they may be used as biological weapons and need to be detected by themselves to warn about an attack to avoid poisoning. For example, the LD50 value of the cholera toxin (CT) is 250 μg/kg in mice,2 but the symptoms such as diarrhea show up at an order of magnitude lower doses.4 Although the lethal doses of staphylococcus enterotoxins are relatively high, its’ sublethal quantity of about 1 μg/person causes notable intoxication syndrome in humans.5 Such a low physiologically active quantity requires extremely sensitive techniques capable of detection of trace amounts of bacterial toxins. Summarizing the facts discussed above, a test-system needed for the detection of biotoxins should be highly sensitive, rapid, and relatively cheap to be accessible for all countries. It would be highly desirable also to provide an opportunity for detection of several toxins simultaneously. In the present work, we focused on development of such an analytical system for the following toxins: cholera toxin (CT) produced by Vibrio cholerae, heat-labile toxin of Escherichia coli (LT), and three

umerous epidemic outbreaks are caused worldwide by water-borne and food-borne gastrointestinal pathogens. The recent example includes the cholera epidemics in Tahiti which killed almost 5000 people.1 Less hazardous, but more widespread bacteria such as staphylococci and E. coli also may produce serious infection outbreaks with many people injured. Besides known hygienic means, such outbreaks could be prevented by a constant monitoring of possible infection sources (drinking water, food, air) for the presence of pathogens. Though in most diagnostics the assay speed is not essential, one can think of many situations where quick assay would be extremely valuable. Thus, quick screening of goods and people for infection biomarkers in airports and other public buildings during epidemics presents examples where rapid assay would be extremely valuable. Rapid diagnostics of infection markers would allow one to immediately intervene in lifethreatening cases in the emergency rooms. One effective and common way to discover bacterial pathogens is the detection of their biomarkers, bacterial toxins, which generally are stable proteins secreted by these bacteria in large quantities.2 Other ways include polymerase chain reaction (PCR)-based methods which discover DNA or RNA bacterial signatures3 and methods which employ biosensors to detect whole bacterial cells.3 Bacterial toxins should also be detected © 2012 American Chemical Society

Received: February 26, 2012 Accepted: June 6, 2012 Published: June 6, 2012 5596

dx.doi.org/10.1021/ac300567f | Anal. Chem. 2012, 84, 5596−5603

Analytical Chemistry

Article

Table 1. Summary of the Detection Methods Described for the Selected Biotoxins method

simultaneously detected analytes

hydrogel-based microarray immunoassay with monoclonal antibodies (Ab)

15

hydrogel-based microarray immunoassay with monoclonal Ab

7

polyclonal Ab microarray with fluorescent nanoparticles detection

3

ELISA with polyclonal Ab ELISA with monoclonal Ab sandwich immunoassay with quantum dot detection

1 1 4

sandwich immunoassay using fluorescent coded microspheres

6

fluoro-immunoassay biosensor

6

immunoassay using fluorescent magnetic beads microsphere-based microarray with signal amplification

5 3

monoclonal Ab microarray

6

piezoelectric immunosensor impedimetric immunosensor immunoassay with ganglioside-liposomes electrochemical immunosensor with ganglioside-liposomes

1 1 1 1

a

Only toxins tested in this research are listed.

biotoxinsa SEA SEB CT LT SEA SEB SEB CT TSST TSST SEB CT SEB CT SEB CT SEB CT SEB CT SEB SEA CT CT CT

LOD, ng/ mL

analysis time

2.3 1.2 0.5 10 0.3 0.5 0.1−0.01

2h

9

2h

10

>3.5 h

11

0.01 0.06 >10

3h >2 h ∼2 h

12 13 14

0.064 1.6 ≥0.5

2.5 h

15

24 min

16

>3.5 h 1.5 h

17 18

>1 h

19

15 min >1 h 20 min >1 h

20 21 6 7

0.003 0.004 0.1 26 412 20 1 10−2−10−5 10−6

references

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EXPERIMENTAL SECTION Materials and Chemicals. The following reagents were purchased from Sigma-Aldrich Co. (St. Louis, MO): bovine serum albumin (BSA), ovalbumin (Ova), cholera toxin (CT), Tween-20, poly(vinyl alcohol) (PVA), poly(vinyl pyrrolidone) (PVP), imidazole (Im), glycine (Gly), Pluronic F-127, trehalose, sodium chloride, sodium borohydride, phosphate buffered saline (PBS) tablets, and Sephadex G-25. Enterotoxin A (SEA) and enterotoxin B (SEB), heat-labile toxin of Escherichia coli (LT), and toxic shock syndrome toxin (TSST) were kindly provided by professor Yu. V. Vertiev from the Clostridiosises laboratory in the Gamaleya Scientific Research Institute of Epidemiology and Microbiology of RAMS (Moscow). Monoclonal antibodies against CT, SEA, SEB, LT, and TSST were prepared, characterization, and biotinylated as described in the Supporting Information section. Dialysis membranes from regenerated cellulose (Fisherbrand, MWCO of 12−14 kDA) were obtained from Fisher Scientific (Pittsburgh, PA). Streptavidin (SA)-coated superparamagnetic beads, 1 μm diameter, Dynal MyOne, were acquired from the Invitrogen (Carlsbad, CA, U.S.A.). The solutions were prepared using deionized Milli-Q water. Sample Preparation. The detection system was first evaluated using solutions of biotoxins in a buffer A which included 0.5% PVA, 0.5% PVP, 0.1% Tween-20, and 20 mM Im titrated to pH 9.3 with solid Gly. The high pH value was chosen so that all the analyzed toxins (having pI values in the range of 6.6−8.8) were negatively charged in the assay conditions. Minced beef was extracted with the PBS buffer (25 mL per 25 g of meat) at room temperature for 10 min. The mixture was then centrifuged at 500g for 10 min, and the aqueous phase was

Staphylococcus aureus toxins: enterotoxins A (SEA) and B (SEB) and toxic shock syndrome toxin (TSST). Several singleanalyte and multiplex test systems reported in the literature for these biotoxins are summarized in Table 1. It is seen that with a few exceptions known techniques take more than 1 h and have limit of detection (LOD) in the range of 0.1−10 ng/mL. Ultrasensitive assays described for CT6,7 became possible due to extremely strong and specific interaction of CT with its unique native ligand, ganglioside GM1.8 Since such ligands are not found for other toxins, a similar approach cannot be applied for them. In the present work, we report a relatively cheap immunoassay method for simultaneous detection of five toxins with a supreme combination of characteristics: multiplexity, low LOD, and brief assay time. The method is based on the approach developed recently22−25 and includes development of additional steps and adaptations to enable simultaneous multiplex analysis. The ultrasensitive “active” immunochemical assay described22 was shown to combine zeptamolar sensitivity with a short analysis time, up to several minutes. These unique characteristics make this technology a very promising tool for development of practically important test systems for detection of various antigens. Some elements of the technology were employed in a rapid ultrasensitive assay of West Nile viruses.23 However, until now, no R/D has been made to adapt such an analytical system for practical multiplex analysis. Here, we report the application of the “active” assay technology for simultaneous multiplex detection of five bacterial toxins: CT, LT, SEA, SEB, and TSST. 5597



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Flow Cell and Peripheral Devices. The detailed description of the flow cell design is given in ref 22. Briefly, it consists of three flow channels: one channel (3) for analyte solution and two others (4) and (5) for electrode buffers, as illustrated schematically in Figure 1. A piece of microarray-1, 3

used in the assay. All analyzed products (water, milk, and meat extract) except for the negative controls were spiked with certain amounts of toxins before preparation of samples. Water samples obtained from the local water-supply system (tap water), a natural pond, and a river were centrifuged for 1 min at 3000g to remove mechanical contaminations. Cow milk samples (with the fat content of 2.5%) were centrifuged for 5 min at 5000g. Both the sediment and the fat on the top were discarded, and the interphase was taken for analysis. Thus, prepared contaminated milk and meat extracts were desalted using a modified centrifugation-assisted gel-filtration technique.26 Sephadex G-25 was washed with 10 volumes of deionized water and 1 mL of gel slurry placed into a column made of pipet tip. A small (100 μL) volume of centrifuged sample was applied to the column and centrifuged for 10 min at 500g. The fraction passed through the column was collected, diluted with 11 μL of the concentrated buffer (200 mM Im titrated with Gly to pH 9.3, 5% PVP, 0.5% Tween-20), and used for assay. Estimation of Fat and Protein Content in the Analyzed Samples. To estimate fat content in our samples, they were extracted with chloroform. The organic phase was collected and dried on a quartz resonator to determine mass of the dry residue as described.27 The latter was attributed to the fat content. The protein concentration in the defatted samples was determined by the Bradford method28 with BSA solutions as standards. Microarray Fabrication. Microarray consisted of nine spots including five spots of monoclonal antibodies against each toxin, three CT spots as positive controls, and one Ova spot as a negative control. All proteins were prepared to arraying by an overnight dialysis against distilled water at 4 °C. After measuring protein concentration by the quartz crystal microbalance technique,25,29 a 10-fold excess of trehalose (w/w) was added to each protein solution; then, it was diluted with water to a final protein concentration of 0.2 mg/mL. Twenty μL aliquots were stored frozen at −25 °C before microarray fabrication. The microarray was fabricated on a plasma-activated dialysis membrane by the electrospray deposition method.25,29 Briefly, the dialysis membrane was treated in a radio frequency plasma discharge in air for 15−20 s at 0.5−1 Torr to introduce aldehyde groups onto the surface for a covalent linking of protein molecules via Schiff bases. A polyester mesh (cat. no. CMY-0150D, Small Parts, Miami Lakes, FL, USA) was used as a mask in the electrospray deposition. Composed of 96 μm thick threads with mesh openings of 150 μm, it allows deposition of array spots, 50 × 50 μm2, spaced by 246 μm.24 After deposition, the microarray was kept for 1 h in a chamber at 100% humidity and 20 °C, and then treated in a solution of sodium borohydride and blocked as described in our recent publication.30 After blocking, the microarrays were stored in 50% glycerol at −20 °C. Binding Biotinylated Antibodies to SA-Coated Magnetic Beads. A suspension of 108 SA-beads (corresponding to 10 μL of the commercial 1% suspension) in 300 μL of PBS buffer containing 1% BSA, 0.1% Tween-20, and 1 μg of biotinylated antibody was incubated under constant mixing for 30 min. Then, 50 μL of a 1 mM water solution of biotin was added, and the mixture was further incubated for 5 min. The beads were then washed 5 times with 0.05% solution of Tween20 in water and stored at 4 °C. Such procedure was applied to each of five biotinylated antibodies.

Figure 1. Cross-section view of the flow cell: (1) microarray, (2) upper membrane, (3) analyte flow cell, (4) upper electrode chamber, (5) lower electrode chamber, (6) O-ring spacer, (7) analyte/beads flow in, (8) analyte/beads flow out, (9) cooling buffer in, (10) cooling buffer out, (11) negative electrode, (12) positive electrode, (13) magnetic concentrator, (14) dark field illuminator, and (15) microscopic lens.

× 15 mm2 (1), is clamped through a spacer (6) to another piece of dialysis membrane (2), forming the analyte channel (3), approximately 100 μm high and 1.2 mm wide, with a microarray facing the channel. The other side of each membrane is facing a separate chamber cooled with a flow of buffer and connected to a loop of platinum wire (11, 12). A nonuniform magnetic field is created by a constant cylinder magnet with a conic concentrator (13) placed underneath the flow cell. Tethering of magnetic beads on the microarray spots is observed under a low-power microscope (15) equipped with a dark field illumination system. Except for the magnet, illuminator (14), and a CCD camera (not shown in the schematic), the device includes a power supply, a pump for cooling electrode buffer, and two syringe pumps for the sample and the suspension of magnetic beads, respectively. Assay Procedure. A schematic of the assay procedure is presented in Figure 2. The sample was pumped through the flow cell for 3 min (Step 1 in Figure 2). During the pumping, a constant voltage (100−150 V, positive electrode under the microarray, negatively charged toxins moving to the microarray surface) was applied to the cell and the latter was cooled by flow of the electrode buffer (20 mM Im titrated with solid Gly to pH 8.5, 0.01% F-127) having a rate of 10 mL/min. The voltage was chosen for each analyte in such a way as to have the resulting heating power below 0.5 W. After passing the sample, a mixture of five biotinylated antibodies which contained 0.2 μg/mL of each antibody in a buffer solution B was pumped through the flow cell for 2 min at a rate of 40 μL/min with the voltage and cooling buffer flow as described above for the sample (Step 2 in Figure 2). The buffer solution B included 0.5% PVA, 0.5% PVP, 0.1% Tween-20, and 20 mM Im titrated to pH 8.5 with solid Gly. After the voltage was switched off, the magnet was immediately placed underneath the flow cell and the suspension of SA-coated magnetic beads (0.001%, w/w, in the buffer A) was pumped through the flow cell at a flow rate of 8−12 μL/min for 2 min (Step 3 in Figure 2). The image was then taken by the CCD camera. Thus, all detection procedures were performed in the same flow cell and included three consecutive stages following 5598



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Keeping the same total beads concentration in the suspension of 5 kinds of beads coated with different antibodies would require reduction of concentration of beads for each specificity by a factor of 5. That would require increasing the scanning time 5-fold to keep the signal level obtained with one sort of bead. Provided that in the latter case the signal appears in ∼2 min of scanning, simultaneous analysis of 5 different analytes would require 10 min. In another approach, antibody molecules of different specificity could be mixed before immobilization on the beads surface. Obviously, thus labeled beads will bind less strongly and less reliably due to a lower number of specific antibodies in the bead-surface contact area. To avoid this limitation, we introduced here an additional active step of binding the secondary antibody molecules labeled with biotin. Since no restriction on the overall concentration of biotinylated antibodies is imposed, cost of multiplexity consists of an increase of the assay time by an additional 2 min only, independent of the number of analytes (within reasonable limits, of course). Now, only one sort of magnetic bead coated with SA is used to detect all the antigen−antibody complexes on the microarray. Another advantage of the protocol presented here consists of a notable gain in the assay sensitivity obtained in the detection of the complex between analyte and the biotinylated antibody with SA-beads as compared with the protocol in which the same biotinylated antibody is first bound to SA-beads and then the beads are used to probe the antigen bound to the antibody on the microarray. The difference in LOD obtained by the two techniques reaches 1−2 orders of magnitude in different cases. Thus, the LOD value was 0.1 pg/mL in the 3-step detection of SEA and SEB but only 1 pg/mL for SEA and 1 μg/mL for SEB upon detection by the 2-step procedure. We speculate that this difference may be attributed to restrictions on the mobility and orientation of immobilized immunoglobulin molecules imposed by the links as well as to random orientation of immobilized molecules making many of them inactive. In contrast, free biotinylated secondary antibody has more opportunity to approach the microarray surface and establish a specific bond with microarray-bound toxin. Finally, being attached to the antigen, it presents several (usually 2−3) biotin molecules for binding to SA-coated magnetic beads which increases the chances for beads tethering. Assay of Biotoxins in Model Solutions and Evaluation of Cross-Reactivity. Protein microarray manufactured by electrospray deposition on the dialysis membrane consists of continuously repeated identical 3 × 3 clusters. Each cluster contains five spots of antibodies plus one negative control (Ova) spot and three positive controls (PC). Schematic of our microarray design is presented in the lower right corner in Figure 3. Though all five toxins should be spotted as PC, we used only cholera toxin spots for simplicity and for unambiguous positioning of other spots with respect to the angle-like structure of three PC spots clearly seen in Figure 3. With the size of microarray cluster being 250 μm, about 50 individual clusters are visible in the flow cell (see Figure 4C) during the assay, making possible a reliable statistical processing of the signal. Though all the antibody pairs used in this work have been evaluated for cross-reactivity in a conventional ELISA format (see Supporting Information for details), we found that their use in the “active” assay was not always successful: from three antibody pairs tested for LT assay, only one was suitable for “active” assay, whereas the other two showed notable cross-

Figure 2. Schematic description of the assay procedure. Step 1: electrophoretical capturing of toxins on a microarray from flow. Step 2: active electrophoretic labeling of the captured analyte by biotinconjugated secondary antibodies. Step 3: visualization of the microarray-bound biotin labels by scanning the microarray surface with streptavidin-coated magnetic beads in a shear flow and magnetic field.

each other with only several second-long intervals. In total, the whole procedure took approximately 7 min. In a two-step procedure, after electrophoretic collection of biotoxins made as described above for the three-step procedure, a suspension of magnetic SA-beads precoated with biotinylated antibodies was pumped through the flow cell to scan the array surface for bound toxins. Due to reduction in the number of steps, this assay procedure may be performed in 5 min. Image Analysis. The microarray images were obtained using a DCM510 USB camera (SkopeTek, China). Each image was manually divided into zones corresponding to the toxinspecific and control spots. The number of tethered magnetic beads on each spot was determined using a home-written software which counted bright spots of a certain size on the dark background. To test the software, the beads were also counted manually and the results were compared. The difference did not exceed 5%. The mean signal (average number of beads on the spot) as well as its standard deviation (STD) was computed for each spot. The signal was considered as positive if the signal/noise ratio (computed as (mean signal − negative control signal)/(STD of the negative control)) exceeded 2.5, which corresponds to ∼0.99 confidence level. The LOD was defined as the minimal analyte concentration giving positive signal in the assay. Safety Considerations. It is recommended for one to place the electrophoretic unit on a nonconductive table and avoid touching its part while running electrophoresis.

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RESULTS AND DISCUSSION Though a general protocol follows the method described earlier, one important novelty was introduced here to improve the assay multiplicity. In the previous protocol, secondary antibodies were directly linked to the scanning magnetic beads.23 That creates a problem when assay multiplexity increases. Mixing beads with different functionalities while keeping the same concentration of each sort will increase the overall concentration of beads which increases their chances for aggregation in the magnetic field within the flow cell.31,32 5599



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The limit of detection (LOD) was determined for each biotoxin dissolved in a low-conductance buffer solution A. The LOD concentrations determined in a series of solutions with toxin concentrations differing by a factor of 10 were found to be of 0.1 pg/mL for SEA, SEB, TSST, and CT and of 1 pg/mL for LT. As seen from Figure 5, in the case of TSST and SEB, a statistically significant signal was visible even at 0.01 pg/mL, but due to variability in the microarray properties (see Figure 4C), the image was not observed on every piece of microarray in a series of identical experiments (positive signal was obtained in 2 cases out of 3 experiments). It was found that the electrophoretic concentrating from flowing analyte solution reduces the LOD by 3 orders of magnitude: detection of CT in the buffer solution A with the electric field switched off resulted in the LOD of 100 pg/mL while a similar procedure with the electric field gave a LOD of 0.1 pg/mL. Despite using antibodies with relatively low affinity (dissociation constants, Kd, in the nanomolar range; see the Supporting Information), we obtained LOD values corresponding to the antigen concentrations of 10−14−10−15 M in our active assay procedure. Such low concentrations are well beyond the reach of the conventional ELISA and other “passive” immunoassay techniques employing the same antibody molecules. Apart from a very short assay time, extremely low LOD originating from preconcentrating of charged analyte in a micrometer−thick layer over the microarray surface24 is the most important advantage of the “active” assay. In fact, in the active assay, no requirement for high affinity of antibodies is imposed. At a low analyte concentration, probability of its binding to antibody is proportional to the ratio of Ceff /(Kd + Ceff), where Ceff is the effective concentration of antibody molecules in the concentrated layer. Because approximately 100 ng/cm2 of active antibody molecules occupies a microarray spot,25 Ceff is ∼10 μM, and since Ceff ≫ Kd for most antibody molecules, any analyte molecule brought into the layer will be bound. The only requirement for the antibody is that the antigen−antibody complex should survive the whole procedure, i.e., the lifetime of the complex must exceed several minutes. As it was mentioned above, antigens are further differentiated not by the thermodynamic stability of the antigen−antibody bond but by its mechanical strength which governs the specificity in the active assay.24 Detection of Toxins Added to Real Samples. Application of the “active” technology is restricted by its requirement for a low electrical conductance of analyzed sample. High conductance does not allow one to use high electric fields in electrophoresis without overheating the solution in the flow cell. The maximum electric power was set in the present design of the flow as ∼1 W. At this power, the solution passing through the flow cell was heated by 5−7 °C. With a conductivity of 11 mS/cm, up to 250 V may be used at a flow rate of 40 μL/min.22 However, practically, we did not use such extreme conditions: samples were desalted to a conductance less than 2 mS/cm. Fresh water from both natural sources (river and pond) and tap water were shown to have the conductivity of 0.5−0.6 mS/ cm and were tested without desalting. Other real samples such as food or seawater have much higher salt concentrations and need desalting to be analyzed by this method. Such a procedure could be performed in 3−4 min using the Quick Spin Protein columns (Roche Diagnostics Corp.), for example. Some of our samples (meat and milk) included complex protein and fat mixtures which requested introduction of additional stages of

Figure 3. Illustration of the assay specificity. In each experiment, 100 μL of each toxin at a concentration of 1 μg/mL was used in the assay. For the viewing clarity, the individual spots are framed and the scheme of the microarray structure is given in the right lower corner. Sign (+) denotes positive control (CT spots); sign (−) denotes negative control (Ova spot), and other spots represent antibodies specific to the indicated toxins.

Figure 4. Illustrations of signals obtained on real samples with different concentrations of toxins added: (A) for the river water and (B) for milk samples. Panel B also includes a schematic structure of the microarray (see capture to Figure 3 for more details). Panel (C) illustrates a general view of the whole microarray visible in the flow cell area (sample: tap water containing 1 pg/mL TSST). Concentration of each of five toxins in the mixture is given at the top of each image. For the viewing clarity, the individual spots are framed.

reactivity with CT, which was not substantial in conventional ELISA. We speculate it is because the requirements for the antibodies to be used in our assay are different from those in traditional “passive” immunoassay.24 Antigen recognition in our method is based on the mechanical strength of the antigen− antibody bond evaluated by a lifetime of the bond under a load applied to the tethered bead in the shear flow, while in the conventional ELISA, antigens are differentiated by their equilibrium binding constants. Though the kinetic dissociation constant correlates well with the thermodynamic dissociation constant, mechanical strength of the bond depends also on many other factors (see the latest review33) which affect the rupture of antigen−antibody bonds. After preliminary tests, certain antibodies were discarded and the final set of arrayed antibodies displayed no cross-reactivity even at a very large concentration of toxins as it is illustrated in Figure 3, where no false positive signals are visible even at concentrations as high as 1 μg/mL for each toxin. These experiments demonstrate the lack of cross-reactivity both between the primary and the secondary biotinylated antibodies since taken in mixture they do not show cross-reactivity to different toxins in the “active” assay format. 5600



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Figure 5. Dependence of signals on concentration of toxins in different media. The error bars both in assay on the same microarray and on different microarrays are nearly equal and correspond to 2.5 × STD. The average positive control signal values were 103 ± 31 beads/spot for buffer solution, 121 ± 38 beads/spot for water samples, 89 ± 33 beads/spot for milk samples, and 96 ± 29 beads/spot for meat extract samples. The background level was 1 ± 1 beads/spot. Letters over each image indicate the toxin tested in each case.

Thus, the detection of every toxin is not affected by the presence of other toxins in the sample. Considering that the protein content in milk and meat was ∼1%, toxins were selectively detected among ∼1010 excess of other protein molecules. The data presented in Figure 5 illustrate that the dynamic range of the method described here spans for about 3 orders of magnitude by concentration. The signals obtained in different media for the same analyte concentration are close to each other (within the confidence intervals); however, the average values are slightly higher in water and in model solution than in milk and meat extract. The signal values at a toxin concentration of 100 pg/mL are nearly equal to the positive control signal indicating full saturation of all binding sites on the spot. It is also seen that, although the average signal grows steadily with the rise in the analyte concentration, the signal confidence intervals are relatively broad and often intersect each other at different toxin concentrations. The most likely reason for this variability is nonuniformity of the microarrays used in assay. As seen in Figure 4C, there is a notable variation in intensity of spots within the observation area. Though partly variations in the intensity could be attributed to a depletion in concentration of toxins at the output of the flow cell due to binding to the microarray, most irregularities originate from variations in the properties of dialysis membrane surface. Gradient of magnetic field which determines the force imposed on the magnetic beads is not uniform, changing by ∼10% over

sample preparation to avoid possible interference of these components with the assay. The initial conductivities of these media after centrifugation were 5.5 and 9.5 mS/cm for the meat and milk samples, respectively; after desalting as described in the Experimental Section, their conductance dropped to 0.4 mS/cm. The protein and fat concentrations were further determined in the desalted samples as 0.5% and 0.02% for the meat extract and 1.5% and 0.5% for the milk. The values of LOD obtained in water samples (from both tap water and water from a local pond and river) were just the same as in the model media described above (∼1 pg/mL for LT and ∼0.1 pg/mL for all other toxins as seen in Figure 5). At the same time, LOD values for all the toxins in the milk and meat extract were ∼1 pg/mL. A few examples of microarray images obtained in the assays of these samples are presented in Figure 4. To determine whether the decrease in the sensitivity resulted from loss of the toxins at the desalting step, we performed control experiments in which the toxins were added to the milk and meat extracts after desalting to exclude the loss of toxins on the gel-filtration column. LOD values in such control samples matched those obtained in water (data not shown), indicating that the toxins were partially lost during the desalting procedure while the “active” immunoassay was not substantially affected by the presence of proteins, fat, and other macromolecular components in the milk and meat extracts. It should also be noted that LODs obtained for simultaneous detection of all five biotoxins coincide with the LOD values of single toxin analysis. 5601



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the scanning area.25 Somewhat higher signal may be obtained (both for specific and control spots) in the areas where gradient of magnetic induction is higher. We should note that irregularities in microarray surface present a significant problem in the “traditional” microarray-based assays as well,34 and similar problems affect our “active” assay technology too. We conclude, from the aforesaid, that in the present form the assay technology described here is suitable only for a qualitative analysis giving positive signal when the analyte concentration exceeds the LOD. For many tasks (like the detection of bacterial pathogens), qualitative character of the assay result may be suitable, considering that a “yes-or-no” result may be obtained quickly, reliably, and with extremely high sensitivity. Our data show that even at toxin concentrations 1 order of magnitude above the LOD the positive signal is reproducibly obtained: no false negative results were registered in 10 independent experiments. Thus, no data prohibiting possible implementation of the described assay in practice were obtained in this research. Of course, using this method to determine precise toxin concentrations requires solving the problem of microarray variability mentioned above.

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CONCLUSIONS It was demonstrated that highly efficient analytical systems can be developed on the basis of ultrasensitive “active” assay technology reported earlier. The assay described here enables simultaneous detection of five bacterial toxins (SEA, SEB, TSST, LT, and CT) with the sensitivity 0.1−1 pg/mL in less than 10 min. Considering the highest toxin molecular weight of ∼80 kDa and the sample volume of 100 μL, the lowest LOD reached corresponds to 100 zeptomoles or 105 toxin molecules in the probe. After a quick conventional sample preparation procedure, samples as simple as water from different sources and as complex as meat and milk can be analyzed with this technique. The technique does not require complex and expensive devices and could be reproduced in an average laboratory having access to mechanical and electronic shops.

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ASSOCIATED CONTENT

S Supporting Information *

Experimental procedures used to prepare and characterize monoclonal antibodies. The photograph of the flow cell assembly on a microscope table. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 7-496-733-0553. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge funding from Russian National Technological Base fund (2007−2011, Grant No. 11411.1003702.13.054 from 06.06.2011). Fruitful discussions with Andrew Mikheev and Igor’ Kanev are gratefully acknowledged. The authors also acknowledge the substantial contribution of Dr. Tamara Morozova to the preparation of the manuscript.

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REFERENCES

(1) Enserink, M. Science 2011, 332, 776−777. 5602



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Rapid Simultaneous Ultrasensitive Immunodetection of Five Bacterial

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