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)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Fundamentos de los sensores yFundamentos de los sensores yFundamentos de los sensores y biosensores químicos ópticosFundamentos de los sensores y biosensores químicos ópticos
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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.
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
(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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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.
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
An optical transducer allowsquantification of:quantification of: AMPLITUDE PHASE FREQUENCY (or )
A FREQUENCY (or ) POLARIZATION KINETICS (impulse)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (5/6)(5/6)OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (5/6)(5/6)WorkingWorking principleprinciple of of thethe transducertransducer (5/6)(5/6)WorkingWorking principleprinciple of of thethe transducertransducer (5/6)(5/6)
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Excitationlight
OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (6/6)(6/6)OpticalOptical sensorssensors::WorkingWorking principleprinciple ofof thethe transducertransducer (6/6)(6/6)WorkingWorking principleprinciple of of thethe transducertransducer (6/6)(6/6)WorkingWorking principleprinciple of of thethe transducertransducer (6/6)(6/6)
66.. REFRACTOMETRYREFRACTOMETRY:sensitive to variations of the refraction index of themedium surrounding the sensor
A. SURFACE PLASMON RESONANCE (SPR)B. INTERFEROMETRY
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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)( )
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
fiber
FIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATION
• 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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
sensitive tip and of the modified radiation to the detector
FIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATIONFIBEROPTIC SENSOR CONFIGURATION
• 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)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
REAGENT PHASE locationREAGENT PHASE locationREAGENT PHASE locationREAGENT PHASE location
A) At the fiber distal end
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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 &
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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.
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
BIOSELECTIVE BIOSELECTIVE LAYERLAYER
BIOSELECTIVE BIOSELECTIVE LAYERLAYER
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
(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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Anticuerpo-antígeno
ANTICUERPOS
AntígenoAntígenomarcado
Anticuerpo anti-analito
Respuesta inversamente
Proporcional a la
TransductorTransductor
Proporcional a la concentración de
analito
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
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
Señal normalizada SN min][)(
A
ICanalitoBB b
50
01
Señal normalizada SN min][)(
A
ICanalitoBB b
50
01
Señal normalizada SN min][)(
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
r 0.998 (n = 5)Moreno-Bondi, Orellana, et al., J. Agric. Food Chem. 2005, 53, 6635
Catedrático de Universidad
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012