Im m u n o a s s a y fo r E s tr o g e n s

in th e E n v ir o n m e n t:
7 6
M u lti- A n a ly te - D e te k tio n b a s e d o n F lu o r e s c e n c e
S . R e d e r1, F . D ie te rle 1, C . B a rz e n 1, I. C o ille 1, R . A b u k n e s h a 2, G . G a u g litz 1

1
In s titu t f r P h y s ik a lis c h e C h e m ie , U n iv e rs ity o f T u e b in g e n , 7 2 0 7 6 T b in g e n , F R G
2
D iv is io n o f L ife S c ie n c e s , K in g s C o lle g e L o n d o n , L o n d o n W 8 7 A H , G B

In tr o d u c tio n S e t-u p fo r T IR F
T h e r e is a r is in g n u m b e r o f s u b s ta n c e s w h ic h v e r ifia b ly (T o ta l In te r n a l R e fle c tio n F lu o r e s c e n c e )
s h o w e s t r o g e n ic a c t iv it y . T h e y c o n ta m in a te th e e n v ir o n -
m e n t, e s p e c ia lly th e s u r fa c e w a te r . A w a t e r m o n it o r in g T h e R iv e r A n a ly s e r ( R IA N A ) is b a s e d o n s p e c ific
s y s t e m is n e e d e d . im m u n o a s s a y s fo r a n a ly te re c o g n itio n a n d T o ta l In te rn a l
R e fle c tio n F lu o r e s c e n c e a s tra n s d u c e r p r in c ip le . T h e d e v ic e
T h e o b je c tiv e is th e e s ta b lis h m e n t o f a n im m u n o a s s a y fo r c o n s is ts o f a n o p tic a l d e t e c tio n u n it, a flo w c e ll, a n d a n
e s tro g e n s in e n v ir o n m e n t. T h e in s tr u m e n t s h o u ld b e s im p le , in t e g r a te d flu id h a n d lin g b a s e d o n flo w in je c tio n ( F IA ) .
lo w - c o s t a n d s m a ll- s iz e d to u s e it fo r th e m o n ito r in g o f w a te r
q u a lity . C o lle c tio n
P D L o c k In
f ib r e s
W e p r e s e n t a m e th o d to d e te c t s e v e r a l e s t r o g e n i c L a s e r F ilte r

s ub s ta n c e s s im u lt a n e o u s ly b y a h e te r o g e n e o u s p h a s e C h o p p e r P C
a b s o rb in g c o a t
im
m u n o a s s a y . A n a ly te s p e c ific a n tib o d ie s a re la b e lle d w it h
a
flu o r e s c e n c e d y e (C y 5 .5 ) a n d re a c t w ith th e a n a ly te in th e B e v e l
W a v e g u id e

s a
m p le . T h e u n o c c u p ie d a n tib o d ie s c a n b in d t o th e m o d ifie d
s u
r fa c e o f a g la s s tr a n s d u c e r. T h e d e te c tio n is b a s e d o n V a lv e
F IA -
S y s te m
T IR F . P u m p

S a m p le s
W a s te

T r a n s d u c e r M o d ific a tio n R e s u lts

s in g le a n a ly te m e a s u r e m e n t
F o r th e s p a tia lly r e s o lv e d s u r fa c e m o d ific a tio n
th e g la s s tr a n s d u c e r is s ila n is e d w ith a n T r a n s d u c e r m o d ifie d w ith a s in g le a n a ly te d e r iv a tiv e
e p o x y s ila n e .
S u b s e q u e n tly a c o n ju g a te o f a m in o d e x tr a n w ith L a b o r a to r y C o n d itio n s : W a s te W a te r S a m p le s :
1 2 0
a n a ly te d e r iv a tiv e is im m o b ilis e d w ith a m ic r o - 1 2 0
S a m p le C o n c e n tra tio n [p p b ]
0 .5 g /m l 1 g /m l w a s te w a te r m e a s u re d tru e
d r o p d is p e n s e r o n p r e - d e fin e d d e te c tio n s p o ts . 1 0 0
A n ti- E s tr o n
1 0 0
A n ti- E s tr o n 1 0 .4 1
C o v a le n t b o n d s e n s u r e a s t a b le s u r fa c e T w e e n
r e l. S ig n a l [% ]

2 2 3
r e l. S ig n a l [ % ]

8 0 8 0
3 9 1 9 0
m o d ific a tio n . 6 0 6 0

4 0 4 0

2 0 2 0

2 - 4 m m 0 0
0 0 .0 1 0 .1 1 1 0 1 0 0 0 0 .0 1 0 .1 1 1 0 1 0 0
E s t r a d io l c o n c e n t r a t io n [ p p b ] E s tr a d io l c o n c e n tr a tio n [p p b ]
Im m o b ilis a t io n w it h a m ic r o d is p e n s in g d e v ic e

d e r iv a tiv e
O
m u ltip le a n a ly te m e a s u r e m e n t
N
a m in o d e x tr a n
O
O H M ix tu r e o f e s tr a d io l a n d e th y n y le s tr a d io l s im u lta n e o u s ly a n a ly s e d
E p o x y s ila n e O N
b in d e d o n g la s s
N
H
O
E s tr a d io l T r u e -P r e d ic te d - P lo t E th y n y le s tr a d io l T r u e -P r e d ic t e d -P lo t
tra n s d u c e r H O H
1 2
S i O M e 3
N H 2
1 0 C a lib r a tio n 1 0 C a lib r a tio n
P r e d ic te d C o n c e n tr a tio n [p p b ]

P r e d ic te d C o n c e n tr a tio n [p p b ]

O O S i O M e 3

O O C r o s s v a lid a tio n C r o s s v a lid a tio n

8 8
T e s t D a ta T e s t D a ta
6
6
4
1 .4 1 .4
4 1 .2
2 1 .2
1 .0 1 .0
0 .8 0 .8
0 .6
0 0 .6

2 0 .4
0 .2
0 .4
0 .2

-2
0 .0 0 .0
- 0 .2 - 0 .2
- 0 .2 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4 - 0 .2 0 .0 0 .2 0 .4 0 .6 0 .8 1 .0 1 .2 1 .4

D a ta A n a ly s is
0 2 4 6 8 1 0 -2 0 2 4 6 8 1 0 1 2
T r u e C o n c e n tr a tio n [p p b ] T r u e C o n c e n tr a tio n [p p b ]

A p p lic a t io n o f a c a lib r a tio n f u n c t io n f o r r e m o v in g

a n y t im e d e p e n d e n c y o f m e a s u r e m e n t s
N o r m a liz a t io n a n d C e n t e r in g a n d S c a lin g o f d a t a
A n a ly s is b y n e u r a l n e t w o r k s : S u m m a r y a n d O u tlo o k
O n e n e u r a l n e tw o r k fo r e a c h a n a ly te T h e lim it o f d e te c t io n fo r E s t r a d io l a s s in g le a n a ly t e is 0 .2 p p b . T h e t e s t m id p o in t is 0 .7 p p b .
A p p lic a tio n o f p r u n in g a lg o r ith m s d u r in g tr a in in g
In d e p e n d e n t d a ta fo r te s tin g th e n e tw o r k s
T IR F a s s a y fo r th e m e a s u r e m e n t in w a s te w a te r g iv e s a g o o d p r e d ic tio n .
T h e a s s a y fo r s im u lta n e o u s d e te c tio n o f tw o a n a ly t e s w it h a c r o s s r e a c tiv e a n tib o d y is
e s t a b lis h e d .
M u lt iv a r ia te d a t a c a n b e a n a ly s e d b y n e u r a l n e tw o r k s . Im p r o v e m e n t w ill b e o b ta in e d b y
e n la r g in g t h e d a t a s e t .
S im u lta n e o u s a s s a y s fo r B is p h e n o l A , E s tr a d io l a n d E th y n y le s t r a d io l w ill b e
e s ta b lis h e d . N e x t s te p is th e m e a s u r e m e n t o f N o n y lp h e n o l a n d C h r y s e n e .

A c k n o w le d g e m e n t
In p u t H id d e n O u tp u t T H E F U N D IN G F R O M T H E E N V IR O N M E N T A N D C L IM A T E P R O G R A M
O F T H E E U IS G R A T E F U L L Y A C K N O W L E D G E D (E N V 4 -C T 9 8 -0 8 0 1 ).
ta n h id e n tity + b ia s