Study and Monitoring of different contaminants in river water
using an Immunosensor
S. Rodriguez-Mozaza, E. Mallata, R. Abukneshac, G. Gauglitzb and D. Barcelóa,
Poster Presentation: 5th Euroconference on Environmental Analytical Chemistry: The impact of (Bio-)Sensors
and Bioanalytical Techniques on Environmental Monitoring. Sept. 8-12th, 2001, College Cork and Vienna
University of Technology, Blarney, Ireland.
a
Dept. of Environmental Chemistry, IIQAB-CSIC, c/ Jordi Girona Salgado 18-26, 08034 Barcelona, Spain
b
Institute of Physical Chemistry, University of Tübingen, 72076 Tübingen, Germany
c
King's College London, University of London, W8 7AH London, England
Environmental legislation has increased the interest in developing analytical devices which
can afford the regular monitoring of organic pollutants in water. As a consequence, biosensors
seems to be a promising tool to obtain fast, sensitive, specific and reproducible measurements.
A solid-phase fluoroimmunoassay combined with an optical transducer to achieve excitation
and collection of fluorescence from fluorescently labeled antibodies locally bound at the
planar interface was developed. The excitation of the fluorophores by an evanescent field can
be adaptable to an optical transducer configuration such as attenuated total internal reflection
element (ATR), which allows the selective detection of surface bound fluorophores.
Determinated compounds are atrazine, simazine, alachlor, isoproturon, 2,4-D and paraquat.
The results obtained with the immunosensor were correlated with classical chromatographic
techniques. The Immunosensor was applied to determine chlorotriazines in river samples
during a monitoring study.
This year the project AWACSS (Automated water analyser computer supported system),
which is a follow-up of RIANA, has started and it will include a new design of the RIANA
prototype with further applications in the environmental area. Different high priority
pollutants like glyphosate, LAS or endocrine disrupters, for example bisphenol A, nonyl and
octylphenol, have been selected to be monitored in this project.
Acknowledgement
This work has been supported by the commission of the European communities, RIANA
(contract
ENV4-CT95-0066)
and
AWACSS
(EVK1-CT-2000-00045)
Study and Monitoring of different contaminants in
river water using an Immunosensor .
S Rodríguez-Mozaza, E, Mallat a, R, Abukneshac, G. Gauglitzb and D. Barcelóa
aDept.
of Environmental Chemistry, CID -CSIC, 08034 Barcelona
of Physical Chemistry, University of Tübingen, 72076 Tübingen, Germany
cKing’s College London , University of London , W8 7AH London, England
bInstitute
The immunosensor RIANA was applied to the trace determination and validation of different contamintants: atrazine, simazine, alachlor, isoproturon, 2,4-D and paraquat. The
inmunosensor system detection mechanism is based on a solid-phase fluoroinmunoassay combined with an optical transducer chemically modified with ananalyte
derivative The results obtained with the immunosensor were correlated with classical chromatographic techniques such as, SPE-LC-MS.
Objectives
Application of the immunosensor to monitor organic contaminants in river water
samples
PD
polymer
fibres
Study the effect of cross-reactants and the matrix on the ìmmunosensor response
Confirmation of the results obtained by the immunosensor using conventional
analytical methods
filter
laser
1 .0 0
Cl
N
N
HN
bevelled
polished end-face
a tr az ine
Cross-reactivity
CH
N
NH
CH 2
N
N
C H3
C H2
HN
AV S ig D iff
Atrazine
N
NH
s im a z in e 0 .1 p p b
0 .7 5
Cl
CH3
CH 3
C H2
C H3
S i m azi n e
(S ta nd a rd IC50
(a bs e nc e o f c ro s s - re a c t a nt )
(S ta nd a rd IC50
(pre s e nc e o f c ros s - re a c t a nt )
)
PC
absorbing
coating
C r o s s - r e a c tiv it ie s
A t r a z in e - S i m a z in e
CH 3
Lock In
s im a zi n e 1 p p b
waveguide
s i m a z i n e 10 p p b
0 .5 0
si m a zi ne 1 00 p pb
inject
FIA System
0 .2 5
C r o s s-r e a c t i v i t y ( % ) =
)
0 .0 0
1 0 -6
1 0 -5 1 0 -4
1 0 -3 1 0 -2
1 0 -1
10 0
10 1
102
samples
10 3
A t r az in e c o n c e n t r a tio n ( p p b )
waste
0.35
0.3
0.25
Regeneration
0.2
Simazine
April 2
immunose nso r LC-M S
n.d.
0.05
n.d.
0.04
n.d.
0.13
1.0
0.92
0.46
0.05
n.d.
1.46
0.97
n.d.
0.04
0.18
0.03
0.03
0.21
0.09
n.d.
0.09
n.d.
0.09
0.04
n.d.
0.22
0.34
0.37
0.41
0.07
0.03
0.71
0.47
May 3
n.d.
0.05
0.37
0.08
0.05
0.42
0.18
June 1
n.d.
0.10
n.d.
0.07
0.03
n.d.
0.20
June 2
n.d.
0.10
0.51
0.05
0.04
0.55
0.19
June 3
n.d.
0.05
0.33
0.02
0
0
Base line
100
200
0.03
0.36
0.10
300
400
500
600
700
800
Incubation
time (s)
Sam p lin g p o in ts:
1 Ebre river
deethylatrazine and total triazines in the Ebre area after
Ebro Delta
total triazines
immunose nso r LC-M S
0.04
May 2
C onc en tra tions ( g/L) of atrazine , simazine ,
Riverine area
Portugal
LC -M S
May 1
April 3
Monitoring study
Deethylatrazine
Atrazine
immunose nso r LC -M S
April 1
0.1
0.05
(  g/L)
Concentratio n
AV
Fluorescence(mV)
0.15
IC50 = Inhibition concentration at 50% of the absorbance
2 Channel
their analysis by SPE -LC -APCI -MS and the
3 Encanyissada
lagoon
immunosensor .
Matrix effect
Atrazine
Simazine
Isoproturon
2,4D
Alaclor
Paraquat
MilliQ Water
IC50
LDD
0.56
0.06
0.33
0.08
1.03
0.01
0.70
0.12
0.27
0.001
0.09
0.01
River Water
IC50
LDD
3.22
0.18
1.21
0.12
1.65
0.14
1.16
0.28
0.47
0.04
017
0.06
Groundwater
IC50
LDD
1.25
0.26
2.79
0.10
1.80
0.17
-
Encanyissada lagoon
Validation
1.00
1.00
(total triazines ( g/L)
1.00
2,4 D (((µg/L)
µg/L)
)
0.75
0.75
0.50
LC-MS
Biosensor
isoproturon (  g/L)
1.25
0.50
0.25
0.25
0.00
0.00
0.25
0.50
0.75
certified isoproturon sample  g/L)
1.00
0.25
0.50
0.75
certified 2,4 D sample (µg/L)
1.00
Results
0.75
Detection limits in river water samples were around 0,1-0,2 μgL-1
0.50
0.25
0.00
0.00
Some overstimation of real values was observed owing to the presence of
cross reactants and matrix effects. No false positives were detected.
0.25 0.50 0.75
1.00 1.25
Biosensor (total triazines (g/L))
1.50
Validation of the immunosensor by SPE-LC-MS, GC-MS and EC-uv
Acknowledgements
This work has been supported by the comission of the European communities , RIANA (ENV-CT95-0066), and AWACSS (EVK1-CT-2000-00045)
RIANA partners: Institute of Physical Chemistry, Univ. Tübingen, Germany; Optoelectronics Research Centre, Univ. Southampton, United Kingdom; Central Research Laboratories,
Hayes Middlesex, Unitd Kingdom; DVGW- Technologiezentrum Wasser, Karlsruhe, Germany; Environmental Intstitute Kos, Slovak Republic; King’s college London , United Kingdom;
Siemens, Erlangen, Germany; CID-CSIC, Barcelona, Spain; Major subcontractor: Catalan Water Agency (Agència Catalana de l’Aigua) Barcelona, Spain,