Sensores ópticos químicos para análisis industrial y ... · Esquema del cursoEsquema del curso...

Sensores ópticos químicos paraSensores ópticos químicos paraSensores ópticos químicos para análisis industrial y control de Sensores ópticos químicos para análisis industrial y control de procesosprocesos

Prof. Dr. Guillermo Prof. Dr. Guillermo Orellana MoraledaOrellana Moraleda

Grupo de Sensores OptoquímicosGrupo de Sensores Optoquímicosy Fotoquímica Aplicada (GSOLFA)

U i id d C l t d M d idUniversidad Complutense de MadridFacultad de Ciencias Químicas (España)

Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012

[email protected] www.ucm.es/info/gsolfa

Esquema del cursoEsquema del cursoEsquema del cursoEsquema del cursoEsquema del cursoEsquema del cursoEsquema del cursoEsquema del curso5ª FEIRA 22/03/2012 6ª FEIRA 23/03/2012

09:00 –11:00

Fundamentos de los sensores y biosensores químicos ópticos.

Cultivos de microalgas para producción de biocombustiblesquímicos ópticos. biocombustibles

11:00 –12:00

Sensores basados en luminiscencia.

Visita a laboratorios del IAPAR

12:00 –14:00

Almuerzo Almuerzo

14:00 – Sensores ópticos Sensores luminiscentes para14:00 15:00

Sensores ópticos químicos para análisis de procesos.

Sensores luminiscentes para monitorización de cultivos de microalgas.

P fé P féPausa‐café Pausa‐café

15:30 –18:00

Prácticas de laboratorio Discusión del CASO PRÁCTICO propuesto (O2)


Fundamentos de los sensores yFundamentos de los sensores yFundamentos de los sensores y biosensores químicos ópticosFundamentos de los sensores y biosensores químicos ópticos


ObjetivosObjetivosObjetivosObjetivosObjetivosObjetivosObjetivosObjetivos

1 C l t d1. Conocer el concepto de sensor.

2. Familiarizarse con las características de los sensores ópticosópticos.

3. Conocer los diferentes principios de medida que permiten desarrollar sensores ópticosdesarrollar sensores ópticos.

4. Las fibras ópticas en los sensores ópticos.

5 Sensores ópticos basados en moléculas indicadoras y sin5. Sensores ópticos basados en moléculas indicadoras y sin indicador (“label‐free”)

6. Biosensores ópticos químicos6. Biosensores ópticos químicos

7. Fundamentos de luminiscencia y fotoquímica para desarrollo de sensores ópticos.


p

“Traditional” vs. modern analysis“Traditional” vs. modern analysis“Traditional” vs. modern analysis“Traditional” vs. modern analysisTraditional vs. modern analysisTraditional vs. modern analysisTraditional vs. modern analysisTraditional vs. modern analysisChr


ris Riddell

Sensors today: The “senses” of electronicsSensors today: The “senses” of electronicsSensors today: The “senses” of electronicsSensors today: The “senses” of electronicsSensors today: The senses of electronicsSensors today: The senses of electronicsSensors today: The senses of electronicsSensors today: The senses of electronics


G. Orellana, in Optical Chemical Sensors, NATO Sci. Ser. Vol. 224, Baldini, Chester, Homola, Martellucci (Eds.), Springer-Kluwer, 2006; pp. 99–116.

“A chemical sensor is a device that transforms chemical information ranging frominformation, ranging from the concentration of a specific sample component to total compositionto total composition analysis, into an analytically useful signal”

(IUPAC 1991)


(IUPAC 1991)

Basics of a chemical (bio)sensorBasics of a chemical (bio)sensor

OPTICALELECTROCHEMICALELECTROCHEMICALELECTRICALMASSTHERMAL

0.000THERMALMAGNETICOTHERS

ANALYTE RECEPTOR TRANSDUCER TRANSMISSION PROCESSINGSTORAGE

ANALYTE RECEPTOR TRANSDUCER TRANSMISSION PROCESSINGSTORAGE


The idea(l) of a chemical sensor…The idea(l) of a chemical sensor…The idea(l) of a chemical sensor…The idea(l) of a chemical sensor…

In situ (at situ)

Continuous (regenerable) Continuous (regenerable)

Real-time (quasi)

Reagent-free (analyzer) Reagent-free (analyzer)


G. Orellana et al., “Online monitoring sensors”, in Treatise on Water Science, vol. 3, P. Wilderer (Ed.), Oxford: Academic Press, 2011; pp 221–262.

F DF

TS

F DF

LIGHTSOURCE

DETECTOR

SOURCE Lamp LED Laser diode

PMT Photodiode PDA CCDFF

Laser

WAVELENGTH

CCD

OPTICALFIBER

FF

“Optrode”“Optode”

SELECTORSFilters (glass, interf.)MonochromatorsG ti

CELL


Gratings

AdvantagesAdvantages of of chemicalchemicalAdvantagesAdvantages of of chemicalchemicalggoptooptosensorssensors

ggoptooptosensorssensors Not subjected to electrical interferences or risks

Contact-less measurements and/or long distance Contact-less measurements and/or long distance

Lack of analyte consumption

"New" analytes may be determined

Easy miniaturization Easy miniaturization

Multiplexing capability and distributed sensing

Higher information density can be transported

Simplicity ruggedness and cost


Simplicity, ruggedness and cost

DrawbacksDrawbacks ofof chemicalchemicalDrawbacksDrawbacks ofof chemicalchemicalDrawbacksDrawbacks of of chemicalchemicaloptooptosensorssensors ((notnot limitinglimiting!)!)DrawbacksDrawbacks of of chemicalchemicaloptooptosensorssensors ((notnot limitinglimiting!)!)

Interference from ambient light

Stability of the indicator phase (photobleaching, leaching, degradation,...)eac g, deg adat o ,...)

Limited dynamic range and response to concentration

Availability of indicator dyes and optoelectronic components.


OpticalOptical sensorssensors accordingaccording totoOpticalOptical sensorssensors accordingaccording totoOpticalOptical sensorssensors accordingaccording totomeasuringmeasuring principleprincipleOpticalOptical sensorssensors accordingaccording totomeasuringmeasuring principleprinciple

ABSORPTION ABSORPTION ABSORPTION ABSORPTION REFLECTANCEREFLECTANCELUMINESCENCE (LUMINESCENCE (flfl // h hh h ))LUMINESCENCE (LUMINESCENCE (fluorescencefluorescence//phosphorescencephosphorescence))CHEMILUMINESCENCECHEMILUMINESCENCERAMAN SCATTERINGRAMAN SCATTERINGREFRACTION INDEXREFRACTION INDEXREFRACTION INDEXREFRACTION INDEX


An optical transducer allowsquantification of:quantification of: AMPLITUDE PHASE FREQUENCY (or )

A FREQUENCY (or ) POLARIZATION KINETICS (impulse)


OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (1/6)(1/6)OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (1/6)(1/6)WorkingWorking principleprinciple of of thethe transducertransducer (1/6)(1/6)WorkingWorking principleprinciple of of thethe transducertransducer (1/6)(1/6)

1.1. ABSORPTIONABSORPTION Absorption of light by the analyte or reagent layer Absorption of light by the analyte or reagent layer Measurements in the UV, VIS, NIR or IR regions of

the electromagnetic spectrum.the electromagnetic spectrum.

Analytical parametersparameters : I, .Io IA = l c

A = i li ci cc

l


l

OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (2/6)(2/6)OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (2/6)(2/6)

22 REFLECTANCEREFLECTANCE for measurements in opaque media

WorkingWorking principleprinciple of of thethe transducertransducer (2/6)(2/6)WorkingWorking principleprinciple of of thethe transducertransducer (2/6)(2/6)

22.. REFLECTANCEREFLECTANCE, for measurements in opaque media,with immobilized indicators.

I id t SPECULAR (“regular reflection”) DIFFUSE:

• Some light absorbed by the particles

Incidentbeam

• Some light absorbed by the particles• Some light scattered by the particlesin all directions Air

MediumKUBELKA-MUNK function

Specular component

U UN u c o(infinite thickness layer):

F(R) = (1-R)2/2R = c/S


Specular component Diffuse component

F(R) (1 R) /2R c/S

OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (3/6)(3/6)OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (3/6)(3/6)

33 LUMINESCENCELUMINESCENCE::

WorkingWorking principleprinciple of of thethe transducertransducer (3/6)(3/6)WorkingWorking principleprinciple of of thethe transducertransducer (3/6)(3/6)

33.. LUMINESCENCELUMINESCENCE:: Fluorescent/phosphorescent ANALYTE (IF = kcA)

A reaction of the ANALYTE with a REAGENT yields a A reaction of the ANALYTE with a REAGENT yields aluminescent PRODUCT (IF = k’cP)

The ANALYTE MODIFIES the light emitted by the The ANALYTE MODIFIES the light emitted by the luminophore: “QUENCHING” (dimming)

STATIC QUENCHINGF + Q FQ

I0/I = 1 + Keq[Q]

F + hF* F + h´

I0/I = 1 + KSV[Q]

K k

DYNAMIC QUENCHING


F + hF* + Q F + Q*

KSV = kQ

Emission lifetimeEmission lifetime--based optical sensors:based optical sensors:PhPh itiiti titi l dl d l i d t til i d t tiEmission lifetimeEmission lifetime--based optical sensors:based optical sensors:PhPh itiiti titi l dl d l i d t til i d t tiPhasePhase--sensitivesensitive vs. vs. timetime--resolvedresolved luminescence detectionluminescence detectionPhasePhase--sensitivesensitive vs. vs. timetime--resolvedresolved luminescence detectionluminescence detection

I = I0 exp(t/

phase (º) timetan = 2f

p ( ) time

Decay time INDEPENDENT of dye concentration, light source intensity and ddetector age: less signal drift, longer sensor lifetime, higher precision

Luminescence profile NOT AFFECTED by static quenching:p y q g extended linear range of calibration curves


More sophisticated instrumentation Dye immobilization leads to non-exponential decays

Optical sensors:Optical sensors:Working principle of the transducer (Working principle of the transducer (4/6)4/6)Optical sensors:Optical sensors:Working principle of the transducer (Working principle of the transducer (4/6)4/6)Working principle of the transducer (Working principle of the transducer (4/6)4/6)Working principle of the transducer (Working principle of the transducer (4/6)4/6)

4. 4. CHEMI/BIOLUMINESCENCECHEMI/BIOLUMINESCENCE: the light emitted by a chemical/biochemical reaction produced in the sensor ( ) i(receptor) element is measured:

A + B C* C* C + h

Low detection limits Reagent consumption

Wide dynamic range Limited scope

Does not require a light source



55 RAMAN SPECTROMETRYRAMAN SPECTROMETRY5. 5. RAMAN SPECTROMETRYRAMAN SPECTROMETRY Involves inelastic scatter of light by molecules

The differences between the excitation light frequency and the Raman bands correspond to the vibrational frequencies of the sample moleculesample molecule

Band intensity depends on the polarizability of the bond

D spectrometerScattered

lightD spectrometer

Excitation


Excitationlight


66.. REFRACTOMETRYREFRACTOMETRY:sensitive to variations of the refraction index of themedium surrounding the sensor

A. SURFACE PLASMON RESONANCE (SPR)B. INTERFEROMETRY


A. SURFACE PLASMON RESONANCE A. SURFACE PLASMON RESONANCE (SPR)(SPR)

PRISM REFLECTEDp-polarizeddi ti

ELIGHTradiation

SP

R = f(,)

SAMPLEMETAL FILM

Au, Ag, Cu, Al, Pt, Ni, Co (50 nm)

SP

, g, , , , , ( )

Coupling between•Photon momentum ENERGY TRANSFER TO

THE METAL SURFACE “PLASMON “PLASMON RESONANCE”RESONANCE”

•Photon momentum•Electron gas momentum

The plasmon wave is sensitive to the refractive index of the sample medium

MEASUREMENTS


At a particular , if we keep a fixed ANGLEAt a particular ANGLE, if we keep a fixed

MEASUREMENTS

SURFACE PLASMON RESONANCE SURFACE PLASMON RESONANCE (SPR)(SPR)

10-3-10-10 M!!!

Dextransupport

Glass wall Gold film


Biacore

B. INTERFEROMETRYB. INTERFEROMETRY SENSITIVEPHASE

DIELECTRICCOATINGPHASE COATING

uo

u1I = I0(1 + cos ) = 2/ L(n - n f)

REFERENCE ARM u u2

2/ L(nsen nref)

L: interaction length

. .Mach-Zehnder interferometer

Based on constructive & destructive wave interference The input light is divided in TWO BRANCHES:

- REFERENCE arm, protected from the surrounding medium- SAMPLE arm SENSIBLE phase

ANALYTEANALYTE modifies refraction indexrefraction index of theANALYTE ANALYTE modifies refraction index refraction index of theCLADDINGCLADDING phasephase

Recombination of light beams


Recombination of light beams change of INTERFERENCE PATTERNINTERFERENCE PATTERN

Core, n1

Buffer & protective layers

1Kao and Hockham suggest in 1966 the use of glass “optical

n1 > n2

fibers” as transmission medium for telecommunications

Cladding, n2

1 2Keck and Schultz (Corning labs) invented the modern optical fiber (1970)(1970)

Guide able to carry the light at Core: physical support of the radiationy g

long distances with minimal losses

radiationCladding: helps confinement of radiation into the fiber


PROPAGATION OF LIGHT WITHIN THE OPTICAL FIBER PROPAGATION OF LIGHT WITHIN THE OPTICAL FIBER (ray theory)(ray theory)PROPAGATION OF LIGHT WITHIN THE OPTICAL FIBER PROPAGATION OF LIGHT WITHIN THE OPTICAL FIBER (ray theory)(ray theory)(ray theory)(ray theory)(ray theory)(ray theory)

IncidentCritical Critical

CCriticalangle C

IncidentReflected

Criticalangle

Refracted

i > c there is transmission by total reflection total reflection

n1 > n2 > n0´n11

n2n0

N A ( 2 2)1/2 /

1 2 0

0


N.A. = sen 0 = (n12 - n2

2)1/2 ; sen C = n2/n10 acceptance angle(maximum angle)

OPTICAL FIBER TYPESOPTICAL FIBER TYPES Silica fibers 200 nm 1.9 m Glass fibers 380 nm 1.9 m Plastic fiber 400 nm 1.2 mIR-transmitting fibers:• Fluoride fibers 1.5 m 4.5 m • Chalcogenide fibers 3 m 11 mg f (As2S3)

• Silver halide fibers 4 m 20 m

(low OH)

(hi h OH)(high OH)


EVANESCENT WAVE EVANESCENT WAVE (latin: evanescer)(latin: evanescer)EVANESCENT WAVE EVANESCENT WAVE (latin: evanescer)(latin: evanescer)

• A small part of the guided light penetrates a tiny distance into the fiber cladding; its intensity decays exponentially from thethe fiber cladding; its intensity decays exponentially from the core/cladding interface (evanescent wave)

• The penetration distance f( n n polarization) is in the• The penetration distance, f(, n1, n2, polarization), is in the order of the propagating radiation wavelength (similar size to many macromolecules, e.g. antibodies).y , g )

n2 EoE

dp

n1 ́

E


FIBEROPTIC SENSOR TYPESFIBEROPTIC SENSOR TYPESFIBEROPTIC SENSOR TYPESFIBEROPTIC SENSOR TYPES

EXTRINSIC SENSORS The sensing element (reagent phase)sensing element (reagent phase), i.e. the component that undergoes modification of its optical properties, is externalexternal to th fibthe fiber

1st GENERATION: The optical change of the analyte itself is measured (PASIVE SENSORS)measured (PASIVE SENSORS) 2nd GENERATION: The optical change of a reagent phase(immobilized indicator) is measured (ACTIVE SENSORS) 3rd GENERATION: Combines the reagent phase with a recognition element of biological origin (e.g. enzyme, antibody, cell,...) (BIOSENSORS)( )


The optical fiber is just a wave guide

INTRINSIC SENSORS The optical fiberoptical fiber itself has the sensing role because one of its propertiesproperties is modifiedmodified.

N h i h i l i f i h h No change in the optical properties of either the analyte or a reagent phase is measured, but the effecteffect of these ones on the propagating lightpropagating light (refraction index of thethese ones on the propagating lightpropagating light (refraction index of the cladding, absorption of light in the evanescent field, interference in FBG, chemically-sensitive core...), y )

Layer with receptor element


The optical fiber is both a wave guide and transducer

FIBEROPTIC SENSING SCHEMESFIBEROPTIC SENSING SCHEMESFIBEROPTIC SENSING SCHEMESFIBEROPTIC SENSING SCHEMES

WAND PROBEFLOW type vsFLOW type vs

PROBE type PROBE type Allow optimization

Easy replacement of h h

FLOW-THROUGH

the reagent phase Accelerate mass

transfer kineticsFLOW THROUGH May be constructed

without optical fiber


FIBEROPTIC SENSING SCHEMESFIBEROPTIC SENSING SCHEMESFIBEROPTIC SENSING SCHEMESFIBEROPTIC SENSING SCHEMES1 Point sensor

Optoelectronics

Measurand field M(t)

FiberSensitive

1. Point sensor

Procesingelectronics

field M(t)Sensitive tip M(t) 2. Distributed

Measurand field M(t)Output M(t) Opto

electronics

Procesing

Fiber

M d fi ld M(t)

3. Quasi-distributed

gelectronics

Output M(t)

M(t)

Optoelectronics

Measurand field M(t)

Fiber

M(z,t)M(t)

zProcesingelectronics

Output M(t)

F r

Sensitized regions


p ( )M(t)

M(z,t)

FIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATION

Si l Smaller size

Lower costSinglefiber

Higher interference input/output light ABSORBANCE

Double Higher

discrimination Doublefiber

input/output light

Collection losses

REFLECTANCEFLUORESCENCE

Singlefiber

FLUORESCENCERAMAN


fiber


• Efficient overlap

Cladding

Fluorescence p

photoexcited sample/fluorescence acceptance cone

Excitation beamCore

acceptance cone• Small size (e.g. catheter)

SAMPLE

• Intrinsic fluorescence and Raman scattering of the fiber

SINGLE FIBER• More difficult separationinput/output light

Simultaneous transmission of the light from the source to the iti ti d f th difi d di ti t th d t t

• Indicator photobleaching


sensitive tip and of the modified radiation to the detector


• Lowers background absorption,

fluorescence and Raman scattering from fluorescence and Raman scattering from the light guide: sensitivity

• Larger size• Less collection efficiency• Critical arreangement of excitation/collection

Dp

Critical arreangement of excitation/collection fibers

• More expensive

Fiber bundle( fi(two fibers

shown)


REAGENT PHASE locationREAGENT PHASE locationREAGENT PHASE locationREAGENT PHASE location

A) At the fiber distal end


REAGENT PHASE locationREAGENT PHASE locationREAGENT PHASE locationREAGENT PHASE location

B) At the fiber sideB) At the fiber siden2n3 n3>n1>n2n1

•Analyte-permeable cladding (technology intensive)

C) In the evanescent fieldC) In the evanescent fieldReagent phase

Hollow waveguide


Hollow waveguide(capillary sensors)

REAGENT PHASE componentsREAGENT PHASE componentsREAGENT PHASE componentsREAGENT PHASE components

“Active” functional groups for immobilizationINDICATOR Keep reactivity towards the analyte

Photostability

Rigid and transparent to light Rigid and transparent to lightSUPPORT High mechanical and bacterial resistance

Favorable analyte-indicator interaction Low reflectance/emission background Low reflectance/emission background

(MEMBRANE) High kinetics of mass transfer Selectivity Isolation from ambient light


Isolation from ambient light

IMMOBILIZATION PROCEDURES (I)IMMOBILIZATION PROCEDURES (I)IMMOBILIZATION PROCEDURES (I)IMMOBILIZATION PROCEDURES (I)

High stabilityg yCOVALENT

ComplexA ti ti f t

AdsorptionPHYSICAL

I l i Activation of supportIndicator functionalizationActive group(s) must remain

Inclusion

FastSimpleSimple

ELECTROSTATIC ReproducibleAccesible active groups

Indicator wash-outIonic strength effects (support &


interaction)

IMMOBILIZATION PROCEDURES (II)IMMOBILIZATION PROCEDURES (II)IMMOBILIZATION PROCEDURES (II)IMMOBILIZATION PROCEDURES (II)

HydrolysisENTRAPMENT: ENTRAPMENT: e.ge.g silicasilica SOLSOL‐‐GEL MATERIALSGEL MATERIALS Hydrolysis

H+

44 ++ H2O (CH3OH)SiOHOH

OHOHSiOCH3

OCH3

OCH3H3CO

Condensation

OHOCH3

+ H2O+ SiOH

OHOH O

OH

OHOHSiSi OH

OH

OHOHSi OH

OH

OHOH

Si OCH3OCH3

OCH3H3CO+ +(CH3OH)Si

OH

OHOH O

OH

OHOHSiSi OH

OH

OHOH

Gelification, aging and drying TimeTemperatureCatalystPore si e


CatalystMorphology

Pore sizeChannel communication

Optochemical sensors:Optochemical sensors:Optochemical sensors:Optochemical sensors:Main application fieldsMain application fields

Process controlProcess control

E i t l l iE i t l l iEnvironmental analysisEnvironmental analysis

Clinical (bio)chemistryClinical (bio)chemistryProf. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012

( ) y( ) y

STAGES OF INDICATORSTAGES OF INDICATOR--BASED SENSOR BASED SENSOR STAGES OF INDICATORSTAGES OF INDICATOR--BASED SENSOR BASED SENSOR

1 SELECTION OF THE COLORIMETRIC OR LUMINESCENT

DEVELOPMENTDEVELOPMENTDEVELOPMENTDEVELOPMENT1. SELECTION OF THE COLORIMETRIC OR LUMINESCENT

INDICADOR WHOSE OPTICAL PROPERTIES ARE MODIFIED IN THE PRESENCE OF ANALYTE.

2. FABRICATION OF THE SENSITIVE PHASE (SUPPORT, MEMBRANE, IMMOBILIZATION PROCEDURE).

3. DESIGN OF THE MOST APPROPRIATE SENSING SCHEME.

4. SPECTROSCOPIC AND ANALYTICAL CHARACTERIZATION OF THE SENSOR.

5. APLICATION AND VALIDATION OF THE OPTODE TO THE TARGET ANALYSIS.


3. Sensor arrays and exchangeable h d Ch t iheads. Chemometrics.

2 C iti f 4. Automatic serial manufacturing Miniaturization 2. Composition of

sensing membranes: New polymer and anti-

manufacturing. Miniaturization, NANOtechnology.Lab-in-a-chip vs. chip-in-a-lab.

1 N i di t d (bi ) h i l

biofouling materials.

1. New indicator dyes, (bio)chemical recognition elements. Molecularly imprinted polymers. Novel transduction


mechanisms (photochemistry).

Un BIOSENSORBIOSENSOR es un dispositivo analítico que incorpora unmaterialmaterial biológicobiológico (e.g. tejido, microorganismo, enzima, gg ( g j , g , ,

anticuerpo, ácido nucleico, etc.) asociado a, o integrado en, un transductor físicoquímico (óptico, electroquímico, térmico,

i lé t i éti )piezoeléctrico o magnético).(Biosens. Bioelectron. 2001, 16:121-131)

BIOSELECTIVE BIOSELECTIVE LAYERLAYER




SignalSignal

LAYERLAYERLAYERLAYER

hh

MembraneExclusion/protection

MembraneExclusion/protection

LAYERLAYERLAYERLAYER

OPTICALOPTICAL

gcapture

andprocessing

Data

OPTICALOPTICAL

gcapture

andprocessing

Datah

ANALYTEANALYTEInterferentsANALYTEANALYTEInterferents

SELECTIVE SELECTIVE ENERGY

TRANSDUCERTRANSDUCER

SIGNAL PROCESSINGENERGY

TRANSDUCERTRANSDUCER

SIGNAL PROCESSING


SAMPLESAMPLESELECTIVE

RECOGNITION EVENT

SELECTIVE RECOGNITION

EVENT

ENERGY TRANSDUCTION

SIGNAL PROCESSINGREADOUT

ENERGY TRANSDUCTION

SIGNAL PROCESSINGREADOUT

Dependiendo de la naturalezanaturaleza de la de la reacciónreacción bioquímicabioquímica de de reconocimientoreconocimiento pueden clasificarse en:

• SENSORES BIOCATALÍTICOS- También denominados sensoressensores metabólicosmetabólicos. . Basados en una

l dl d bb ll llA

reacción catalizadacatalizada que origina un cambiocambio en la en la naturalezanaturaleza químicaquímicade las especies involucradas (analito y otras).

Biológico: enzimas aisladas células tejidosBiológico: enzimas aisladas, células, tejidos(Biomimético: Polímeros de impronta molecular (MIPs))

• SENSORES DE BIOAFINIDAD

B

• SENSORES DE BIOAFINIDAD- Basados en interaccionesinteracciones específicasespecíficas entre el analito y el bioreceptor.

No hay conversión del analito.ABiológico: anticuerpo, ácidos nucleicos (DNA/RNA),

receptores(Bi i éti P lí i t l l (MIP ) li étid li l ótid


(Biomimético: Polímeros impronta molecular (MIPs), oligopétidos, oligonucleótidos, aptámeros)

PRINCIPIO DE MEDIDA ANALITO ENZIMA

(A) El analito se convierte en un PRODUCTO con propiedades ÓPTICAS características (o

FENOL (O2) PPO(Tirox.)

INDUCE un cambio en una señal óptica: pH, desactivación fluor.,…).

(B) V i ió d l i d d (B) Variación de las propiedades ÓPTICAS de la ENZIMA en su

reacción con el analitoLACTATO LMO

(C) El analito INHIBE una reacción enzimática que produce/consume un producto con propiedades ÓPTICAS

PESTICIDAS AChEproducto con propiedades ÓPTICAS


O2 Desactivación Oxidasa Glucosa,colinalluminiscencia colesterol

Lactato, fenolNH3 Variación pH Hidrolasa UreaNH3 Variación pH Hidrolasa UreapH Variación pH pH Glucosa, urea

penicilinasppesticidas

CO2 Variación pH Descarboxilasa Glutamato

TRANSDUCTORBiocatalizador

SUS PROEnzima

Muestra


SUS PRO Muestra

EJEMPLO: pesticidasEJEMPLO: pesticidas

Acetilcolina + H2O Ác. Acético + ColinaAChE

Inhibidores de la AChe:

Transductor químico de Transductor químico de pHpH

Carbamatos Organofosforados

Indicador de tipo ftaleína

Intensa absorción en el visibleCl

HO OH

Elevada estabilidad fotoquímica

Inmovilización relativamente sencillaa través de los grupos fenólicos

Cl

O a través de los grupos fenólicos

pKa = 4.8 (amarillo) ‐ 6.4 (violeta)S

OO

ROJO DE CLOROFENOL


Biosens. Bioelectron. 2000, 14, 895

ROJO DE CLORO ENOL

N

NOCOCH3

NN

Ru2+

emmax = 610 nmem = 0.02= 0.58 s

Inhibidores de la AChe: Carbamatos

OrganofosforadosN N

N

0.58 s

H2O AChEDD

Inhibidor Substrato ENZIMABuffer

NOH

max 695NN NN

N

Ru2+ em

max = 695 nmem = 0.002= 0.15 s


Orellana et al. Afinidad 2007, 64, 257–264

ANTICUERPOS

Zona de unión del antígeno16 nm

Zona de unión del antigeno

Región

variable

Cadena ligera

CH1Región

Región variable de la cadena ligeraVH

variable

Región

12 n

m

CH1

C

VL

Región variable de la cadena ligera

C

Región constante de la cadena ligera

Región

Fab

Región

constante

CH2

CH2

CL constante de la cadena pesada

Fc

Cadena pesada

Heterodímeros con cuatro cadenas polipeptídicas: dos cadenaspesadas idénticas y dos cadenas ligeras idénticas


Fuente: Dr. M.P. Marco (CSIC) Barcelona, Spain

ANTICUERPOS

Interacciones hidrofóbicas

Enlace de hidrógenoEnlace de hidrógeno

Fuerzas de van der Waals

Interacciones electrostáticas

Anticuerpo antígeno


Anticuerpo-antígeno

ANTICUERPOS

AntígenoAntígenomarcado

Anticuerpo anti-analito

Respuesta inversamente

Proporcional a la

TransductorTransductor

Proporcional a la concentración de

analito


Detección óptica directa (sin trazador). i lé l i i l ñ l ó i La propia molécula origina la señal óptica.

Ensayo en fase heterogeneaCompetitivoCompetitivo

No competitivo

Detección óptica indirecta.pSe requiere un trazador o reacciones acopladas

para originar el efecto óptico. Ensayo en fase heterogenea

CompetitivoNo competitivo


p

H

OH

H HO

N

S CH3

CH

NH H H

OOS CHN

H H HO

Penicilina GPenicilina G A i iliA i ili

N

S

COOH

CH3

CH3

NH

O

H H

NH2

Penicilina VPenicilina V

N

COOH

CH3O

ON

S

COOH

CH3

CH3

N

O

Penicilina GPenicilina G AmoxicilinaAmoxicilina

OH H

OH HH

O

A i iliA i ili

N

S

COOH

CH3

CH3

NH

O

H H

NH2N

S

COOH

CH3

CH3O

H HNH

NO CH3

CloxacilinaCloxacilina

N

S

COOH

CH3

CH3O

H HNH

NO CH3

Cl

AmpicilinaAmpicilinaOxacilinaOxacilina

ClO

CloxacilinaCloxacilina

N

S

COOH

CH3

CH3O

H HNH

NO CH3

Cl

O

N

S CH3

CH3O

H HNH

O

OC2H5


DicloxacilinaDicloxacilinaCOOH

NafcilinaNafcilinaCOOH

O

DERIVADOS DEL PIRENODERIVADOS DEL PIRENO

S CHNH H H

O

DERIVADOS DEL DANSILODERIVADOS DEL DANSILO

H HHN

COOH

CH3

CH3OPAAPPAAP ( )3

N

S CH3

CH3

NH

O

H HO

PBAPPBAP DAPDAP

N

S

COOH

CH3

CH3O

NH

SO O

N

COOHOPBAPPBAP

N

S CH3

CH3

NH H H

NH

O DAPDAP

COOHO

O PAAMPAAM

N

S CH3

CH

NH H H

NH

O

( ) N

S CH3

CH

NH H H

NH

O

ON

COOH

CH3O

NH

O

( )3

PBAMPBAMOH

O DAMDAM

N

COOH

CH3O

NHS

OO

N

S CH3

CH3

NH

O

H H

NH

O

N


COOHO

OPAAXPAAX

G. Orellana et al. Patente ES 2197811; Anal. Chem. 2006

Curva de calibradoCurva de calibrado1.21.21.21.2

Ecuación de ajusteEcuación de ajuste

Señal normalizada = S = iminmax)( AAABB

Señal normalizada = S = i

minmax)( AAABB

Señal normalizada = S = iminmax)( AAABB

Señal normalizada = S = i

minmax)( AAABB

lizad

a (S

N)

0.8

1.0

lizad

a (S

N)

0.8

1.0

lizad

a (S

N)

0.8

1.0

lizad

a (S

N)

0.8

1.0

Señal normalizada SN min][)(

A

ICanalitoBB b

50

01


A

ICanalitoBB b

50

01


A

ICanalitoBB b

50

01


A

ICanalitoBB b

50

01

Seña

l nor

mal

0 2

0.4

0.6

Seña

l nor

mal

0 2

0.4

0.6

Seña

l nor

mal

0 2

0.4

0.6

Seña

l nor

mal

0 2

0.4

0.6

[PENG], g mL-1

0.000001 0.0001 0.001 0.01 0.1 1 10 1000.0

0.2

0.0

[PENG], g mL-1

0.000001 0.0001 0.001 0.01 0.1 1 10 1000.0

0.2

0.0

[PENG], g mL-1

0.000001 0.0001 0.001 0.01 0.1 1 10 1000.0

0.2

0.0

[PENG], g mL-1

0.000001 0.0001 0.001 0.01 0.1 1 10 1000.0

0.2

0.0

IC50 30 ng mL-1

Características Analíticas

ID (6.02 – 191) ng mL-1

LD 2.4 ng mL-1

C50 30 ng mL


r 0.998 (n = 5)Moreno-Bondi, Orellana, et al., J. Agric. Food Chem. 2005, 53, 6635

Catedrático de Universidad


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