1Fibre Optic Biosensor for Detection of Drugs of Abuse

Frank F. BierFraunhofer-Institut fr Biomedizinische Technik (IBMT)Institutsteil PotsdamundUniversitt PotsdamInstitut fr Biochemie und BiologieScience Park, Potsdam, Germany

2Outline

biosensors

immunoassays

fibre optic sensors evanescent wave

fluorescence label

experimental setup

some results progesteron in blood and milk

thc in urine

3Outline

biosensors

immunoassays

fibre optic sensors evanescent wave

fluorescence label

experimental setup

some results progesteron in blood and milk

thc in urine

4Basic Principle of Biosensors

Biosensors are measuring devices consisting of

a close and functional contact of aMolecular Recognition Element(Receptor) with an optical or electronical Transducer, (e.g. an electrode or an optical wave guide).

high selectivity even in crude samples e.g. blood, cell extracts, food, soil

Advantages

may be miniaturized: low amount of sample and reagents

5Biosensors

Two types:

Catalysis, Metabolism

Binding

ES P

e-

Y Y

EnzymesRibozymesCells

Electrodes

Antibodies, LectinsNucleic acids (Aptamers, DNA)Molecular Imprints (MIPs)

Quartz MicrobalanceSPRFluorescence fibre optics

6Biosensors

Catalysis, Metabolism

Binding

ES P

e-

Y Y

Electrodes: amperometirc or potentiometric Signal is instantaneous

Any Transducer: (Quartz Microbalance, SPRFluorescence fibre optics, etc.):Signal develops: binding curve, depending on affinity

time

sign

al

7Outline

biosensors

immunoassays (binding assays)

fibre optic sensors evanescent wave

fluorescence label

experimental setup

some results progesteron in blood and milk

thc in urine

8Antibody

9Image credit NIAID: www.niaid.nih.gov/topics/HIVAIDS/Research/Pages/B12antibodyimage.aspx

Epitope mappingParatope mapping

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Immunoassay

Sandwich type enzyme linked immunosorbent assay (ELISA)

Y Y Ycapture antibody probing antibody

Y Y Y

add 2nd Ab, remove, add substrate

Y Y Y

x x

eeee e

Microtiter plateDetection by absorption

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Y Y Ycapture antibody probing antibody

Y Y Y

add 2nd Ab, remove

Immunoassay

Sandwich type fluorescence* immunoassay*or other label

fluorescence measurement black microtiter plates,slides

picture by Heiko Andresen Fraunhofer IBMT

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Principle of an indirect competitive assay - Binding Inhibition Assay

2 31DetectionPre-incubation

time to be definedReaction on the Chip

defined period of incubation

Immunoassay principles

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ProteinProtein--ELISA CellELISA Cell--ELISAELISA DNADNA--HybridisationHybridisation PeptidePeptide--Assay competitiveAssay competitive

surface for capture molecule coupling

capture antibody

labeledprobing antibody

Analyte

capture probe

labeled DNA-probe

Microorganism

DNA/RNA

Molecular Recognition is Basis of Bioanalysis

peptide

Serum antibody hapten

immob.competitor

14

Functionalization of Glass Slides and Fibres

Clean up (acidic wash, organic solvents)

NaOH Silanization

hydrolysis of ethoxy groups

hydogen bond forming between silanols

15

Covalent coupling

16

e.g.. epitope mapping using peptid-arrayAnti-TSH-Receptor ScreeningTSH-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

Immobilisation: molecular orientation

Immobilisation small molecules by use of ProteinsPre-incubation of s.m. with streptavidin equilibration of phys.-chem. differences all s.m. in the same buffer regular droplests, regular spots

17

Heiko Andresen, Kim Zarse et al. 2005

buffer

serum

Assay PerformanceAssay performance depends on mediumPeptide analysis for serology of viral infections

18

Immunoassays are highly specific crossreaction

CH3

O

O OH

OH

OH

O

O

OH

Progesteron C21H30O2 MW: 314,5Pregn-4-en-3,20-dion

Testosteron C19H28O2 MW: 288,44-Androsten-17-ol-3-on17Hydroxy-4-androsten-3-on

stradiol 1,3,5 stratrien-3,17-diol3,17_Dihydroxy-1,3,5(10)-stratrien

C18H24O2 MW: 272,4

Dehydroepiandrosteron (DHEA) C19H28O2 MW:288,4trans-DehydroandrosteronDehydroisoandrosteron5-Androsten-3-ol-17-on

heparinisierte 20 l Kapillare

plus 80 l Reagenz

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

Example: A set of steroid hormones and derivatives

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Hormone- and Hapten-Immunoassays

Example Multiple Parameter Analysis: Fertility Hormone Assays

Analyte Medical range Achieved detection limit (ELISA)

d.l. n n

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

all assays may be performed in multiplex format with low cross reactivity

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Hapten immunoassay with amperometric readout

Readout Systems and Biosensors

21

hCG ELISA with dual-mode readout (amperometry/fluorescence)

New perspectives for immunoassay development

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 antibody-HRP conjugate 2 g mL-1 (30min), Amplex Red 50 mol L-1

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

Dual-mode readout

22

Novel Readout System: Quenching Redox-Mediator Assay

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

- progesteron

+ progesteron

-14

-

11

-8

n

A /

I

0 20 40 60 80 100 c / progesterone [ng/mL]

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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

The parameters that define assay performance

24

Outline

biosensors

immunoassays

fibre optic sensors evanescent wave

fluorescence label

experimental setup

some results progesteron in blood and milk

thc in urine

25

E

x

glass (n = n1)

critical angle c of total internal reflection:

c = arcsin (n2/n1)

Total internal reflection the evanescent field

1

Air or aqueous solution (n = n2)

Extension of evanescent field d (1/e-value):

0 / 2d = n1sin1 - n2

26

Waveguide

27

absorbed powerPabs = P0 - Ptrans P0: input power (excitation)

= P0 (1-e-eff l ) Ptrans: transmitted power

eff : effective absorption coefficient

emitted powerPem = QPabs Q: quantum yield of

fluorescence dye

detected powerPdet = f Pem f: geometry faktor

P0 Ptrans

Pem

Pem

camera/detector

l

waveguide

Efficiency of fluorescence detection

28

wave guide: intrinsic transducer

using the evanescent wave

~ 400 nm~ 20 nm

Surface Plasmon Resonance (SPR) Grating Coupler

29

Fiber optic with evanescent field:

collecting fluorescence only at the surface

DetectorPMT

F

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Experimenteller Aufbau des faseroptischen Sensors

Sensorkopf mit optischer Faser und Fliezelle, vor und nach ThermostatisierungAbt. Mikosysteme/Sensorsysteme

Experimental Setup

Licht aus der unmittelbaren Umgebung der Faseroberflche kann nicht-klassisch einkoppeln

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Fibre optic biosensor with fluorimetric detection ultra-sensitive and quantitative readout (nmol L-1)

real-time detection

disposable fibre optic sensor

flow-through fibre optic biosensor 50 m

m

0 2 4 6 8 10 12 14 16 18 20 220

1

2

3

4

buffersignal

Sig

nal [

Vol

t]t [min]

antibody (2,5 g mL-1)

buffer NaOH 0.1 mol L-1

real-time readout (Cy5-labeled anti-progesterone antibody)

Readout Systems and Biosensors

32

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)

automated fibre optic immunosensor prototype

disposable fibre optic immuno-sensor(for 100 measurements)

33

time / min

Sig

nal /

a.u

.Regeneration

Biosensors multiple use including calibration

example: THC 1,5 15 ng/mL

34

Determination of THC

35

First experiments: Proof of principle

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

36

Immunoassay with second antibody

not useful

Faser-Versuch Telo-Horst Dronnabinol-Komp. an THC-COOH Faser

maximaler Signalanstieg

0

0,5

1

1,5

2

2,5

1500 375 94 23 5,9 1,5 0,4 0,1

Dronabinol [ng/ml]

rFI [

Vol

t]

37

log (THC conc.) (ng/ml)re

l. F

luor

esce

nce

/ nor

m.

5,00 g/ml2.50 g/ml1,25 g/ml0,62 g/ml

Antiboy conc.

Optimization procedure: Titration of sensitivity

direct labelling

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

Sig

nal a

mpl

.out

/ m

V

38

Commercial Analysis Device for Multiple Use

Application for milk analysis (progesteron) at point of care

39

Original Data THC in Urine Samples

measurement in the Milk-machine

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Urin - Kontrollen

Negativ (O ng/ml))

Pos (150 ng/ml)

Mix mit 37,5 ng/ml

Mix mit 75 ng/ml

Mix mit 112,45 ng/ml

PBS

THC Std.reihe

10 g/ml

1 g/ml

100 ng/ml

100 ng/ml

10 ng/ml

nur aMDy

THC Immunoassay in MTP/Slide-Format

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Original Data: THC measurement on routine platform

assay performance depends on antibody quality

high dissoc. rate const.

THC-conc. Signal ()100 ng/ml 1,310 ng/ml 1,51 ng/ml 1,60 ng/ml 1,9

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Spiked Samples: THC in Urine

43

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

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Thanks

Support

Eva Ehrentreich-Frster, Stefan Kubick Doreen WstenhagenMarkus von Nickisch-Rosenegk, Nenad Gajovic-Eichelmann, Heiko Andresen* Ralph Hlzel, Edda Rei Michael Breitenstein,Jutta Ettlinger, Alexander Christmann, Xenia Marschan*,Peter M. Schmidt*, Jrg Henkel, Dennie Andresen,Matthias Griessner, Jenny Steffen*, Frank Kleinjung*, Michaela Schellhase, Dirk Michel, Thomas Nagel*,Claus Duschl, Michale Kirschbaum, Bettina Junker, Christine Miler, Ines Zerbe, Kathi Gromann, Rothin Strehlow, Christian Heise*, Martina Beysel,Andr Lehmann.Jrg Nestler, Thomas Otto, ENASEric Nebling, ISiTAlbrecht Brandenburg, IPMAndreas Teichert, IPAAchim Weber, IGB

F.W. Scheller, U. Wollenberger, University of Potsdam D. Zahn, BST BioSensorTechnologies GmbH, BerlinB. Danielsson, U Lund , SM. Willander, N. Calander, Chalmers, Gothenburg, S P.E. Nielsen, Univ. Copenhagen, DKK. Misiakos, S. Kakabakos, CRNS Demokritos, GRV.A. Erdmann, J. Kurreck, FU Berlin C. Niemeyer, Univ. DortmundH.-R. Glatt, W. Meinl, DIfE, Bergholz-RehbrckeM. Bienert, A. Ehrlich, FMP Berlin-BuchE. Matthes, A. Lehmann, MDC Berlin-BuchH. Heidecke, Celltrend GmbH, LuckenwaldeM. Kuhn, congen GmbH, Berlin-Buch

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End

46

Summary

Biosensors

Thank you for your attention!