Fibre Optic Biosensor for Detection

of Drugs of Abuse

Frank F. Bier
Fraunhofer-Institut fr Biomedizinische Technik (IBMT)
Institutsteil Potsdam
und
Universitt Potsdam
Institut fr Biochemie und Biologie
Science Park, Potsdam, Germany

Outline
biosensors
immunoassays
fibre optic sensors
evanescent wave
fluorescence label
experimental setup

some results
progesteron in blood and milk
thc in urine

Outline
biosensors
immunoassays
fibre optic sensors
evanescent wave
fluorescence label
experimental setup

some results
progesteron in blood and milk
thc in urine

Basic Principle of Biosensors

Biosensors are measuring devices
consisting of
a close and functional contact of a
Molecular Recognition Element
(Receptor) with an optical or
electronical Transducer,
(e.g. an electrode or an optical wave guide).

Advantages
high selectivity even in crude samples
e.g. blood, cell extracts, food, soil
may be miniaturized:
low amount of sample and reagents

Biosensors
Two types:
Catalysis, Metabolism
P

S
E

e-

Binding

Enzymes
Ribozymes
Cells
Electrodes

Antibodies, Lectins
Nucleic acids (Aptamers, DNA)
Molecular Imprints (MIPs)

Quartz Microbalance
SPR
Fluorescence fibre optics

Biosensors

Catalysis, Metabolism
P

S
E

e-

Electrodes: amperometirc or potentiometric

Signal is instantaneous

Any Transducer:

signal

Binding
(Quartz Microbalance, SPR
Fluorescence fibre optics, etc.):

Signal develops: binding curve,

depending on affinity
time

Outline
biosensors
immunoassays (binding assays)
fibre optic sensors
evanescent wave
fluorescence label
experimental setup

some results
progesteron in blood and milk
thc in urine

Antibody

Epitope mapping
Paratope mapping

Image credit NIAID: www.niaid.nih.gov/topics/HIVAIDS/Research/Pages/B12antibodyimage.aspx

Immunoassay
Sandwich type enzyme linked immunosorbent assay (ELISA)
add 2nd Ab,

e
e
YYY
capture antibody

remove,

YYY

add substrate
x
x

e
YYY

probing antibody

Microtiter plate
Detection by absorption

10

Immunoassay
Sandwich type fluorescence* immunoassay
*or other label

add 2nd Ab,

YYY
capture antibody

remove

YYY
probing antibody

fluorescence measurement
black microtiter plates,
slides
picture by Heiko Andresen Fraunhofer IBMT

11

Immunoassay principles
Principle of an indirect competitive assay - Binding Inhibition Assay

1
Pre-incubation
time to be defined

2
Reaction on the Chip
defined period of incubation

3
Detection

12

Molecular Recognition is Basis of Bioanalysis

ProteinProtein-ELISA

CellCell-ELISA

DNADNA-Hybridisation PeptidePeptide-Assay

competitive

labeled DNA-probe

labeled
probing antibody

DNA/RNA
Analyte
capture antibody

Microorganism

Serum
antibody
capture probe
peptide

hapten
immob.
competitor

surface for capture molecule coupling

13

Functionalization of Glass Slides and Fibres

hydrolysis of ethoxy groups

hydogen bond forming

between silanols

Clean up
(acidic wash,
organic solvents)

NaOH
Silanization

14

Covalent coupling

15

Immobilisation: molecular orientation

Immobilisation small molecules by use of

Proteins
Pre-incubation of s.m. with streptavidin
equilibration of phys.-chem. differences
all s.m. in the same buffer
regular droplests, regular spots

e.g.. epitope mapping using peptidarray

Anti-TSH-Receptor Screening
TSH-Rezeptor in 256 Peptiden als Array von
15meren reprsentiert alle Amionsuren

Mit: Charit Berlin (Dr. C. Grtzinger)

Heiko Andresen, Kim Zarse et al. Proteomics 2005

16

Assay Performance
Assay performance depends on medium
Peptide analysis for serology of viral infections

buffer

serum

Heiko Andresen, Kim Zarse et al. 2005

17

Immunoassays are highly specific crossreaction

Immunoassays work in crude samples: Blood, milk, urine etc.

Example: A set of steroid hormones and derivatives

O

OH

CH3

Progesteron C21H30O2 MW: 314,5

OH

1,3,5 stratrien-3,17-diol
3,17_Dihydroxy-1,3,5(10)-stratrien

Pregn-4-en-3,20-dion

heparinisierte 20 l Kapillare

stradiol C18H24O2 MW: 272,4

OH

OH

Testosteron C19H28O2 MW: 288,4

4-Androsten-17-ol-3-on
17Hydroxy-4-androsten-3-on

Dehydroepiandrosteron (DHEA)
C19H28O2 MW:288,4
trans-Dehydroandrosteron
Dehydroisoandrosteron
5-Androsten-3-ol-17-on

plus 80 l Reagenz

18

Hormone- and Hapten-Immunoassays

Example Multiple Parameter Analysis: Fertility Hormone Assays
all assays may be performed in multiplex format with low cross reactivity
Analyte

Medical range

Achieved detection limit (ELISA)

d.l.

Progesterone

0,2 1,5 ng/ml

0,01 ng/ml

0,035

200

Testosteron

0,1 2 ng/ml

0,1 ng/ml

0,036

51

Estradiol

10 - 300 pg/ml

10pg/ml

0,106

21

FSH

2-100 IU/l

2 IU/l

0,078

15

LH

1 - 25 IU /l

1 IU/l

0,046

18

Prolactine

2- 100 g/l

500 ng/ml

0,064

30

SHBG

20 - 120 nmol /l

25 nmol/l

0,046

35

DHEAS

1 - 6 g/ml

1g/ml

0,081

21

TSH

0,2 - 10 IU /l

0,2 IU

0,032

100

hCG

2-1000 IU /l

10 IU/l

0,063

27

19

Readout Systems and Biosensors

Hapten immunoassay with amperometric readout

20

Dual-mode readout
hCG ELISA with dual-mode readout (amperometry/fluorescence)
New perspectives for immunoassay development

Direct amperometric detection of resorufin

in a thin-layer flow cell
(glassy carbon, -0.17 V vs. Ag/AgCl,
buffer pH 5, 0.1 ml min-1 )

Competitive hCG immunoassay in blood serum (detection

limit 30 mU mL-1, incubation in 96-well micro-titer plate
200L, amperometric flow-through detection, n=4)
(PBS , anti hCG 0.05 g mL-1 (60min), secondary antibodyHRP conjugate 2 g mL-1 (30min), Amplex Red 50 mol L-1

21

Novel Readout System: Quenching Redox-Mediator Assay

-14

-11

-8

+ progesteron

nA / I

- progesteron

0
Julia Ettlinger, Nenad Gajovic-Eichelmann, Frank Bier: Biosensors 2010

20 40 60 80 100
c / progesterone [ng/mL]

22

The parameters that define assay performance

the antigen: paratope-epitope interface

interaction constants
kind of immobilization
density of immobilization
label
label efficiency
QC: how reliable are the binding patterns and the binding constants?

ELISA

Peptide
microarray

23

Outline
biosensors
immunoassays
fibre optic sensors
evanescent wave
fluorescence label
experimental setup

some results
progesteron in blood and milk
thc in urine

24

Total internal reflection the evanescent field

E

Air or aqueous
solution (n = n2)

glass (n = n1)

x
critical angle c of total internal reflection: Extension of evanescent field d (1/e-value):
c = arcsin (n2/n1)

d=

0 / 2
n1sin1 - n2

25

Waveguide

26

Efficiency of fluorescence detection

absorbed power
Pabs = P0 - Ptrans
= P0 (1-e-eff l )

waveguide
P0

Pem
l

Ptrans
Pem

camera/
detector

emitted power
Pem = QPabs

detected power
Pdet = f Pem

P0: input power (excitation)

Ptrans: transmitted power
eff : effective absorption
coefficient
Q: quantum yield of
fluorescence dye

f: geometry faktor

27

wave guide: intrinsic transducer

using the evanescent wave
~ 20 nm
~ 400 nm

Surface Plasmon Resonance (SPR)

Grating Coupler
28

Fiber optic with evanescent field:

collecting fluorescence only at the surface

Detector
PMT

29

Experimental Setup
Experimenteller Aufbau des faseroptischen Sensors

Licht aus der unmittelbaren Umgebung

der Faseroberflche kann nicht-klassisch einkoppeln

Sensorkopf mit optischer Faser und Fliezelle,

vor und nach Thermostatisierung
Abt. Mikosysteme/Sensorsysteme

30

Readout Systems and Biosensors

Fibre optic biosensor with fluorimetric detection
ultra-sensitive and quantitative readout (nmol L-1)
real-time detection
disposable fibre optic sensor
4
-1

NaOH 0.1 mol L

Signal [Volt]

50 mm

buffer
3

2
antibody
-1
(2,5 g mL )
1

buffer

signal

0
0

10

12

14

16

18

20

22

t [min]

flow-through fibre optic biosensor

real-time readout (Cy5-labeled

anti-progesterone antibody)

31

Fibre optic biosensor

progesterone in milk (commercialized in 2008)
drugs of abuse screening
online-monitoring of chemical synthesis
kinetic measurement
activity of telomerase (tumor marker)

disposable fibre optic immuno-sensor

(for 100 measurements)

automated fibre optic

immunosensor prototype

32

Biosensors multiple use including calibration

example: THC 1,5 15 ng/mL

Signal / a.u.

Regeneration

time / min

33

Determination of THC

34

First experiments: Proof of principle

TSH-BSA as competitor (analyte) proving the conjugate
sensitive range : 0,1 - 10 ng /mL

35

Immunoassay with second antibody

not useful
Faser-Versuch T elo-Horst
Dronnabinol-Komp. an THC-COOH Faser
maximaler Signalanstieg
2,5

rFI [Volt]

2
1,5
1
0,5
0
1500

375

94

23

5,9

1,5

0,4

0,1

Dronabinol [ng/ml]

36

Optimization procedure: Titration of sensitivity

Antiboy conc.
5,00 g/ml
2.50 g/ml
1,25 g/ml
0,62 g/ml

rel. Fluorescence / norm.

Signal ampl.out / mV

direct labelling

log (THC conc.) (ng/ml)

decreasing
decreasing antibody
antibody conc.
conc. >>.
>>. decreasing
decreasing detection
detection range
range

37

Commercial Analysis Device for Multiple Use

Application for milk analysis (progesteron) at point of care

38

Original Data THC in Urine Samples

measurement in the Milk-machine

39

THC Immunoassay in MTP/Slide-Format

THC Std.reihe

Urin - Kontrollen

10 g/ml

Negativ (O ng/ml))

1 g/ml

Pos (150 ng/ml)

100 ng/ml

Mix mit 37,5 ng/ml

100 ng/ml

Mix mit 75 ng/ml

Mix mit 112,45 ng/ml

10 ng/ml

nur aMDy
PBS

40

Original Data: THC measurement on routine platform

assay performance depends on antibody quality

high dissoc. rate const.

THC-conc.
100 ng/ml
10 ng/ml
1 ng/ml
0 ng/ml

Signal ()
1,3
1,5
1,6
1,9

41

Spiked Samples: THC in Urine

42

Summary
Fluorescence fibre optic sensor
pesticides in water samples
aptamers for explosives
DNA hybridisation
hormones in blood and milk
THC in urine
semi-disposable: the fibre core
disposable: reagents (confectioned)
detector with autosampler

43

Thanks
Eva Ehrentreich-Frster,
Markus von Nickisch-Rosenegk,
Ralph Hlzel,
Jutta Ettlinger,
Peter M. Schmidt*,
Matthias Griessner,
Michaela Schellhase,
Claus Duschl,
Christine Miler,
Rothin Strehlow,
Andr Lehmann.
Jrg Nestler, Thomas Otto, ENAS
Eric Nebling, ISiT
Albrecht Brandenburg, IPM
Andreas Teichert, IPA
Achim Weber, IGB
Support

Stefan Kubick
Nenad Gajovic-Eichelmann,
Edda Rei
Alexander Christmann,
Jrg Henkel,
Jenny Steffen*,
Dirk Michel,
Michale Kirschbaum,
Ines Zerbe,
Christian Heise*,

Doreen Wstenhagen
Heiko Andresen*
Michael Breitenstein,
Xenia Marschan*,
Dennie Andresen,
Frank Kleinjung*,
Thomas Nagel*,
Bettina Junker,
Kathi Gromann,
Martina Beysel,

F.W. Scheller, U. Wollenberger, University of Potsdam

D. Zahn, BST BioSensorTechnologies GmbH, Berlin
B. Danielsson, U Lund , S
M. Willander, N. Calander, Chalmers, Gothenburg, S
P.E. Nielsen, Univ. Copenhagen, DK
K. Misiakos, S. Kakabakos, CRNS Demokritos, GR
V.A. Erdmann, J. Kurreck, FU Berlin
C. Niemeyer, Univ. Dortmund
H.-R. Glatt, W. Meinl, DIfE, Bergholz-Rehbrcke
M. Bienert, A. Ehrlich, FMP Berlin-Buch
E. Matthes, A. Lehmann, MDC Berlin-Buch
H. Heidecke, Celltrend GmbH, Luckenwalde
M. Kuhn, congen GmbH, Berlin-Buch

44

End

45

Summary

Thank you for your attention!

Biosensors

46