1. FIELD PORTABLE IMMUNOSENSORS THAT MEASURE LOW MOLECULAR WEIGHT CONTAMINANTS IN ENVIRONMENTAL WATER SAMPLES Scott J. Melton 1, Ruquia B. Ahmed-Schofield 2, Robert C. Blake II 3, Diane A. Blake 4 1 Interdisciplinary Program in Molecular and Cellular Biology and 4 Department of Biochemistry, Tulane Univ. Hlth. Sci. Ctr. New Orleans LA, 70112 2 Department of Chemistry and 3 Division of Basic Pharmaceutical Sciences, Xavier University of Louisiana, New Orleans, LA 70125 Tel: (504) 988-2478; email: blake@tulane.edu, smelton@tulane.edublake@tulane.edusmelton@tulane.edu Abstract Immunosensors that employed a “kinetic exclusion” method have been used to develop a variety of assays for low molecular weight contaminants. The prototype sensors and associated software were provided by Sapidyne Instruments Inc (Boise, ID). These sensors produced assays that were 100- to 1000-fold more sensitive than competitive microwell-based immunoassays prepared using identical primary antibodies and analyte- conjugates. Analysis of operator-prepared samples in the field-portable instrument required 165 seconds, and CV’s were 10-15%. The in-line sensor had the ability to collect a sample from a process line, add reagents, inject the incubation mixture and compare the results to an instrument-generated standard curve. Total analysis time, including washing and mixing, was between 600 and 900 seconds, and CV’s were 5% or less. These sensors have been used to develop assays for heavy metals and caffeine in environmental and food samples. We are also in the process of developing monoclonal antibodies to brevetoxin selected for optimal performance in these immunosensors. These monoclonal antibodies will be used to develop a field portable assay for brevetoxin that will provide results in a nearly real-time format. Figure 1. Principle Behind the Immunosensor Assay. A. Fluorescently labeled antibody is mixed with ligand (L) and allowed to come to equilibrium. B. The mixture is then passed over beads containing bound ligand. Excess antibody that did not bind to ligand in the first step is captured by the beads. C. As more antibody binds to the column, fluorescent signal increases. The resultant slopes generated are inversely proportional to the amount of ligand present in the incubation mixture. D. A plot of slope versus ligand concentration generates a standard curve that can be used to assess the concentration of the ligand in an unknown sample (1,2). IC 50 =1.25 μM MDL=0.49 μM IC 50 =0.45 nM MDL=0.1 nM cELISA Kinetic Exclusion Figure 2. Comparison of a cELISA with a kinetic exclusion-based assay for caffeine. A. Competitive ELISA for Caffeine. Because caffeine concentrations are relatively high (~30 nM) in water contaminated with human waste, caffeine has been proposed as an anthropogenic marker for untreated wastewater. A competitive ELISA developed in our lab using commercially available reagents (anti-caffeine and a caffeine-BSA conjugate) provided an assay with a minimum detection limit of 500 nM. B. Kineitc Exclusion Assay for Caffeine. This assay used the same anti-caffeine antibody and caffeine-BSA conjugate employed in the cELISA. Because of the very short contact time of the antibody with the caffeine- BSA coated beads, the antibody bound to soluble caffeine is does not have time to dissociate and re-bind to the caffeine-BSA conjugate. This “kinetic exclusion” results in an assay with a limit of detection approximately 4900-fold more sensitive than the cELISA. A B A B Figure 3. Two kinetic exclusion based instruments useful for field-based studies. A. An automated sensor that has the ability to autonomously run a standard curve from stock reagents and to prepare and analyze environmental samples (3). B. A beta prototype field-portable device. Samples are injected over a flow cell containing microbeads coated with ligand-conjugate complex. After the generation of a standard curve, data can be obtained within minutes. The device interfaces with a laptop computer through a wireless connection and weighs less than 12 kilograms. Both instruments promise a wide range of utility for the near real- time analysis of environmental samples Figure 4. Results from field test of the in-line instrument at Oak Ridge National Lab (ORNL). The in-line device shown in Panel A above was transported to ORNL and set up in the laboratories there. After signal tests to insure that the sensor was not damaged during transport, the instrument was loaded with reagents. The sensor autonomously mixed samples with known quantities of uranium (VI) (0-21 nM) to generate a standard curve (∆), then mixed two samples from a bioremediated site with reagents (buffer, chelator, an antibody that recognized a chelated form of U(VI) and Cy5-labeled anti-mouse Fab) for comparison to the instrument- generated standard curve. Purified Water Caffeine ppb spiked inAmount Recovered 1nM0.911nM 91.10% 2nM2.284nM 114.20% 6nM5.578nM 92.96% Average 99.42% 50% Environmental Water Caffeine ppb spiked inAmount Recovered 1nM0.943(SD.173) 94.30% 2nM1.94 (SD 0.095) 97.00% 6nM4.98 (SD.225) 83.00% Average 91.43% Table 1. Results from a caffeine spike and recovery using an alpha prototype of the field portable instrument. Conclusions These data demonstrate that the format of an immunoassay may significantly influence its performance characteristics, even when identical reagents are employed. Immunosensors based on the principal of kinetic exclusion appear to be uniquely suited for the analysis of low molecular weight contaminants. The total time required for sample mixing, incubation and analysis is 5-10 minutes. Modifying the sensor for analysis of a new analyte is as easy as changing the beads in the flow cell and changing the antibody in the incubation mixture. Kinetic exclusion assays have been formulated for caffeine and heavy metals in our laboratory (3,4) and for PAH’s and TNT in aquatic samples at sites near Virginia Institute of Marine Science (5). Assays for marine algal toxins are in development at Tulane. These sensors are expected to fill an important niche in analysis of low molecular weight environmental contaminants. Figure 5. Analysis of caffeine in the field-portable sensor. Soluble caffeine (0-6 nM) was mixed with buffer, anti-caffeine antibody and Cy5-labeled anti-mouse Fab. After 10 minutes at room temperature, the reaction mixture was applied to a flow cell containing beads coated with caffeine-BSA. Delta fluorescence (calculated by subtracting the average the signal from seconds 5-10 of the trace from the signal at 155- 160 seconds of the trace) was plotted versus caffeine concentration. The figure compares the dose- response for caffeine spiked into reagent grade water (red) and into a sample that contained 50% of a caffeine-free environmental water sample (green). Please contact us to discuss development of additional assays for these biosensors The in-line sensor is suitable for deployment in a buoy or jetty. The field portable sensor is ideal for use in a boat or along the shore. Both sensors provide near real-time data about contaminant levels. We are actively trying to identify new marker compounds that could assist investigators in the study of the processes that lead to hypoxia. The ideal marker for these sensors would be a compound that has a molecular weight >400, is structurally complex, and is difficult to measure with current technologies. It should be present in environmental samples at concentrations of at least 10 nM. References 1. Blake, D.A. et al. (1996) Metal binding properties of a monoclonal antibody directed towards metal-chelate complexes. J. Biol. Chem. 271:27677-27685. 2. Blake, R.C. II et al. (1999) Automated kinetic exclusion assays to quantify protein binding interactions in homogeneous solution. Anal. Biochem. Anal. Biochem. 272:123-134. 3. Yu, H. et al. (2005) An immunosensor for autonomous in-line detection of heavy metals: Validation for hexavalent uranium. Int. J. Env. Anal. Chem. 85:817. 4. Blake, D.A. et al. (2001) Antibody-based sensors for heavy metal ions. Biosens. Bioelectron., 16:799-809. 5. Bromage, E.S. et al. (2007) The development of a real-time biosensor for the detection of trace levels of trinitrotoluene in aquatic environments. Biosens. Bioelectron, in press.