FIELD PORTABLE IMMUNOSENSORS THAT MEASURE LOW MOLECULAR WEIGHT CONTAMINANTS
IN ENVIRONMENTAL WATER SAMPLES
1
Scott J. Melton1, Ruquia B. Ahmed-Schofield2, Robert C. Blake II3, Diane A. Blake4
Interdisciplinary Program in Molecular and Cellular Biology and 4Department of Biochemistry, Tulane Univ. Hlth. Sci. Ctr. New Orleans LA, 70112
2Department of Chemistry and 3Division of Basic Pharmaceutical Sciences , Xavier University of Louisiana, New Orleans, LA 70125
Tel: (504) 988-2478; email: blake@tulane.edu, smelton@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 analyteconjugates. 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.
Table 1. Results from a caffeine spike and recovery using an alpha prototype of
the field portable instrument.
B
A
Purified Water
Caffeine ppb spiked in
1nM
2nM
6nM
Average
50% Environmental Water
Caffeine ppb spiked in
1nM
2nM
6nM
Y
Y
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).
RELATIVE SLOPE
fluorescence, volts
Y Y
1
2
3
4
5
6
1.0
0.5
•The total time required for sample mixing, incubation and analysis is 5-10 minutes.
0.80
St andar d Cur ve
0.20
5
10
15
20
25
•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.
Uranium, nM
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 instrumentgenerated standard curve.
Kinetic
Exclusion
IC50=1.25 μM
MDL=0.49 μM
1.10
0.90
0.70
0.50
3
4
5
6
7
8
9
10
100
purified water
IC50=0.45 nM
MDL=0.1 nM
50% Enviromental water
0.40
0.10
0
Caffeine, µM
1
2
5
References
10
[Caffeine], nM
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 caffeineBSA 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.
•The field portable sensor is ideal for use in a boat or along the shore.
•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.
0.30
0.20
0
•The in-line sensor is suitable for deployment in a buoy or jetty.
•We are actively trying to identify new marker compounds that could assist investigators
in the study of the processes that lead to hypoxia.
0.50
50
Please contact us to discuss development of additional assays for these
biosensors
•Both sensors provide near real-time data about contaminant levels.
B
Delta (V)
A
D elta Fluorescence, V
Ab so r b an ce (450-650 n m)
I CP- M S value
0.40
0
cELISA
2
I n- line value
0.60
0
1.50
1
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.
1.00
ANALYTE
CONCENTRATION
0
91.43%
0
time, seconds
1.30
94.30%
97.00%
83.00%
Conclusions
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 realtime analysis of environmental samples
Delta
YY
Y
Y
91.10%
114.20%
92.96%
99.42%
Amount Recovered
0.943(SD .173)
1.94 (SD 0.095)
4.98 (SD .225)
Average
Figure 3. Two kinetic exclusion based instruments useful for field-based studies.
Y
Y
Amount Recovered
0.911nM
2.284nM
5.578nM
0.00
0
2
4
6
8
10
Caffeine Conc entration (nM)
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 155160 seconds of the trace) was plotted versus caffeine concentration. The figure compares the doseresponse for caffeine spiked into reagent grade water (red) and into a sample that contained 50% of a
caffeine-free environmental water sample (green).
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.