Sensores
Sensores ópticos químicos para ópticos químicos para
análisis industrial y control de procesos
Prof. Dr. Guillermo Orellana Moraleda
Grupo de Sensores Optoquímicos
y Fotoquímica Aplicada (GSOLFA)
U i
Universidad
id d Complutense
C
l t
de
d M
Madrid
d id
Facultad de Ciencias Químicas (España)
[email protected]
www.ucm.es/info/gsolfa
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Esquema del curso
5ª FEIRA 22/03/2012
6ª FEIRA 23/03/2012
09:00 – Fundamentos de los 11:00
sensores y biosensores químicos ópticos.
químicos ópticos.
11:00 – Sensores basados en 12:00
luminiscencia.
Cultivos de microalgas para producción de biocombustibles
Visita a laboratorios del IAPAR
12:00 – Almuerzo
14:00
Almuerzo
14:00 – Sensores
14:00
Sensores ópticos ópticos
15:00
químicos para análisis de procesos.
P
Pausa‐café
fé
Sensores luminiscentes para Sensores
luminiscentes para
monitorización de cultivos de microalgas.
P
Pausa‐café
fé
15:30 – Prácticas de laboratorio Discusión del CASO 18:00
PRÁCTICO propuesto (O2)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Fundamentos de los sensores y
Fundamentos de los sensores y biosensores químicos ópticos
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Objetivos
1. C
1
Conocer el concepto de sensor.
l
t d
2. Familiarizarse con las características de los sensores ópticos.
ópticos
3. Conocer los diferentes principios de medida que permiten desarrollar sensores ópticos
desarrollar sensores ópticos.
4. Las fibras ópticas en los sensores ópticos.
5 Sensores ópticos basados en moléculas indicadoras y sin 5.
Sensores ópticos basados en moléculas indicadoras y sin
indicador (“label‐free”)
6. Biosensores ópticos químicos
Biosensores ópticos químicos
7. Fundamentos de luminiscencia y fotoquímica para desarrollo de sensores ópticos.
p
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
“Traditional”
Traditional vs. modern analysis
Chrris Riddell
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Sensors today: The “senses”
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.
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
“A chemical sensor is a device that
transforms chemical
information ranging from
information,
the concentration of a
specific sample component
to 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
Basics of a chemical (bio)sensor
OPTICAL
ELECTROCHEMICAL
ELECTRICAL
MASS
THERMAL
MAGNETIC
OTHERS
ANALYTE
RECEPTOR
TRANSDUCER
0.000
TRANSMISSION PROCESSING
STORAGE
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
The idea(l) of a chemical sensor…
 In situ (at situ)
 Continuous (regenerable)
 Real-time (quasi)
 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.
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
F
F
D
TS

LIGHT
SOURCE
DETECTOR
 Lamp
 LED
 Laser diode
 Laser
WAVELENGTH
SELECTORS
Filters (glass, interf.)
Monochromators
G ti
Gratings
F
OPTICAL
FIBER
 PMT
 Photodiode
 PDA
 CCD

“Optrode”
CELL
“Optode”
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Advantages
g of chemical
optosensors
opto
sensors
 Not subjected to electrical interferences or risks
 Contact-less measurements and/or long distance
 Lack of analyte consumption
 "New" analytes may be determined
 Easy miniaturization
 Multiplexing capability and distributed sensing
 Higher information density can be transported
 Simplicity,
Simplicity ruggedness and cost
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Drawbacks of chemical
optosensors
opto
sensors (not limiting
limiting!)
!)
 Interference from ambient light
 Stability of the indicator phase (photobleaching,
leaching,
eac g, deg
degradation,...)
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
Optical sensors according to
measuring principle
 ABSORPTION
 REFLECTANCE
 LUMINESCENCE (fluorescence
((fl
fl
fluorescence/
/phosphorescence)
phosphorescence
h
h
)
 CHEMILUMINESCENCE
 RAMAN SCATTERING
 REFRACTION INDEX
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
An optical transducer allows
quantification of:
 AMPLITUDE
 PHASE
A
 FREQUENCY (or )
 POLARIZATION
 KINETICS (impulse)


Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Optical sensors:
sensors:
Working principle of the transducer (1/6)
1.
ABSORPTION
 Absorption of light by the analyte or reagent layer
 Measurements in the UV, VIS, NIR or IR regions of
the electromagnetic spectrum.

Analytical parameters : I, .
A=lc
A = i li ci
Io
I
c
l
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Optical sensors:
sensors:
Working principle of the transducer (2/6)
2. REFLECTANCE,
REFLECTANCE for measurements in opaque media,
media
with immobilized indicators.
 SPECULAR (“regular reflection”)
 DIFFUSE:
• Some light absorbed by the particles
• Some light scattered by the particles
in all directions
IIncident
id t
beam




Air
Medium
KUBELKA-MUNK
U
UN function
u c o
(infinite thickness layer):
F(R) = (1
(1-R)
R)2/2R = c/S
 Specular component
 Diffuse component
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Optical sensors:
sensors:
Working principle of the transducer (3/6)
3. LUMINESCENCE
LUMINESCENCE::
 Fluorescent/phosphorescent ANALYTE
(IF = kcA)
 A reaction of the ANALYTE with a REAGENT yields a
luminescent PRODUCT
(IF = k’cP)
 The ANALYTE MODIFIES the light emitted by the
luminophore: “QUENCHING” (dimming)
 STATIC QUENCHING
F+Q
FQ
I0/I = 1 + Keq[Q]
 DYNAMIC QUENCHING
I0/I = 1 + KSV[Q]
F + h
F*  F + h´
F* + Q  F + Q*
KSV =  kQ
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Emission lifetimelifetime-based optical sensors:
Ph
PhasePhase
-sensitive
iti vs. time
ti -resolved
timel d luminescence
l i
detection
d t ti
I = I0 exp(t/
tan  = 2f

phase ((º))
p
time
 Decay time INDEPENDENT of dye concentration, light source intensity and
detector age:
 less signal drift, longer sensor lifetime, higher precision
 Luminescence p
profile NOT AFFECTED by
y static q
quenching:
g
 extended linear range of calibration curves

 More sophisticated instrumentation
 Dye immobilization leads to non-exponential decays
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Optical sensors:
Working principle of the transducer (4/6)
(4/6)
4. CHEMI/BIOLUMINESCENCE
CHEMI/BIOLUMINESCENCE: the light emitted by a
chemical/biochemical reaction produced in the sensor
(
(receptor)
) element iis 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
Optical sensors:
sensors:
Working principle of the transducer (5/6)
5 RAMAN SPECTROMETRY
5.
 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 molecule
 Band intensity depends on the polarizability of the bond
D
spectrometer
Scattered
light
Excitation
light
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Optical sensors:
sensors:
Working principle of the transducer (6/6)
6. REFRACTOMETRY
REFRACTOMETRY:
sensitive to variations of the refraction index of the
medium 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 (SPR)
E
p-polarized
di ti
radiation
PRISM
REFLECTED
LIGHT
R = f(,)
SP
SAMPLE
METAL FILM
Au,, Ag,
g, Cu,, Al,, Pt,, Ni,, Co (50
( nm))
Coupling between
•Photon momentum
•Electron gas momentum
ENERGY TRANSFER TO
THE METAL SURFACE
“PLASMON
RESONANCE”
The plasmon wave is sensitive to the refractive index of the sample medium
MEASUREMENTS  At a particular , if we keep a fixed ANGLE
 At a particular ANGLE, if we keep a fixed 
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
SURFACE PLASMON RESONANCE (SPR)
10-3-10-10 M!!!
Dextran
support
Glass wall Gold film
Biacore
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
B. INTERFEROMETRY
I = I0(1 + cos )
 = 2/ L(nsen- nreff)
L: interaction length
SENSITIVE
PHASE
DIELECTRIC
COATING
u1
uo
u
u2
REFERENCE ARM
.
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
ANALYTE  modifies refraction index of the
CLADDING  phase
 Recombination of light beams
 change of INTERFERENCE PATTERN
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Buffer & protective layers
Core, n1
Kao and Hockham suggest in
1966 the use of glass “optical
fibers” as transmission medium
for telecommunications
Keck and Schultz (Corning labs)
invented the modern optical fiber
(1970)
Guide able to carryy the light
g at
long distances with minimal
losses
n1 > n2
Cladding, n2
Core: physical support of the
radiation
Cladding: 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
(ray theory)
Incident
Critical
angle
Critical
angle
C
C
Reflected
Incident
Refracted
i > c there is transmission by total reflection
n0
n2
n1
n1 > n2 > n0
´
1
0
0
acceptance angle
(maximum angle)
N A = sen 0 = (n
N.A.
( 12 - n22)1/2 ; sen C = n2/n
/ 1
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
OPTICAL FIBER TYPES
 Silica fibers
 Glass fibers
 Plastic fiber
IR-transmitting fibers:
• Fluoride fibers
• Chalcogenide
g
fibers
f
(As2S3)
• Silver halide fibers
200 nm    1.9 m
380 nm    1.9 m
400 nm    1.2 m
1.5 m    4.5 m
3
m    11 
m
4 m    20 m
(low OH)
(hi h OH)
(high
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
EVANESCENT WAVE (latin: evanescer)
• A small part of the guided light penetrates a tiny distance into
the fiber cladding; its intensity decays exponentially from the
core/cladding interface (evanescent wave)
• The penetration distance,
distance f(,
f( n1, n2, polarization),
polarization) is in the
order of the propagating radiation wavelength (similar size to
manyy macromolecules,, e.g.
g antibodies).
)
n2
n1
dp

Eo
E
´
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
FIBEROPTIC SENSOR TYPES
EXTRINSIC SENSORS
 The sensing element (reagent phase),
phase) i.e. the component that
undergoes modification of its optical properties, is external to
th fib
the
fiber
 1st GENERATION: The optical change of the analyte itself is
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
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
INTRINSIC SENSORS
 The optical fiber itself has the sensing role because one
of its properties is modified
modified.
 No
N change
h
iin the
h optical
i l properties
i off either
i h the
h
analyte or a reagent phase is measured, but the effect of
these ones on the propagating light (refraction index of the
cladding, absorption of light in the evanescent field, interference
core...))
in FBG,, chemically-sensitive
y
Layer with receptor element
The optical fiber is both a wave guide and transducer
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
FIBEROPTIC SENSING SCHEMES
WAND PROBE
FLOW type vs
PROBE type
 Allow optimization
 Easy replacement of
the
h reagent phase
h
 Accelerate mass
FLOW-THROUGH
FLOW
THROUGH
transfer kinetics
 May be constructed
without optical fiber
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
FIBEROPTIC SENSING SCHEMES
1 Point sensor
1.
Opto
electronics
Fiber
Sensitive
tip M(t)
Procesing
electronics
Measurand
field M(t)
Output M(t)
2. Distributed
Opto
electronics
Measurand field M(t)
Fiber
Procesing
g
electronics
3. Quasi-distributed
Output M(t)
M
Measurand
d fi
field
ld M(t)
Opto
electronics
Procesing
electronics
M(t)
Output
p M(t)
( )
M(t)
M(z,t)
Fiberr
F
z
Sensitized regions
M(z,t)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
FIBEROPTIC SENSOR CONFIGURATION
 Smaller size
Lower cost
 Higher interference
input/output light
Single
Si l
fiber
ABSORBANCE
 Higher
discrimination
input/output light
Double
fiber
 Collection losses
Single
fiber
REFLECTANCE
FLUORESCENCE
RAMAN
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
FIBEROPTIC SENSOR CONFIGURATION
Fluorescence
Cladding
Core


• Efficient overlapp
photoexcited
sample/fluorescence
acceptance cone
• Small size (e.g. catheter)
Excitation beam
SAMPLE

SINGLE FIBER
• Intrinsic fluorescence and
Raman scattering of the
fiber
• More difficult separation
input/output light
• Indicator photobleaching
Simultaneous transmission of the light from the source to the
sensitive
iti tip
ti and
d off the
th modified
difi d radiation
di ti to
t th
the d
detector
t t
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
FIBEROPTIC SENSOR CONFIGURATION
Dp


•

• Larger size
• Less collection efficiency
• Critical arreangement of excitation/collection
•
Lowers background absorption,
fluorescence and Raman scattering from
the light guide:  sensitivity
fibers
More expensive
Fiber bundle
(
(two
fi
fibers
shown)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
REAGENT 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 location
B) At the fiber side
n2
n3 n3>n1>n2
n1
•Analyte-permeable cladding
(technology intensive)
C) In the evanescent field
Reagent phase
Hollow waveguide
(capillary sensors)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
REAGENT PHASE components
 “Active” functional groups for immobilization
INDICATOR  Keep reactivity towards the analyte
 Photostability

SUPPORT 


(MEMBRANE)
Rigid and transparent to light
High mechanical and bacterial resistance
Favorable analyte-indicator interaction
Low reflectance/emission background
 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
IMMOBILIZATION PROCEDURES (I)
 High
g stabilityy
Adsorption
COVALENT
 Complex
PHYSICAL
Activation
A
ti ti off supportt
Indicator functionalization
Active group(s) must remain
I l i
Inclusion
 Fast
ELECTROSTATIC
Simple
Reproducible
Accesible active groups
 Indicator wash-out
Ionic strength effects (support &
interaction)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
IMMOBILIZATION PROCEDURES (II)
ENTRAPMENT: e.g
ENTRAPMENT: e.g silica SOL‐
SOL‐GEL MATERIALS
 Hydrolysis
OCH3
H3CO Si OCH3 + 4 H2O
OCH3
H+
OH
OH SiOH + 4 (CH3OH)
OH
 Condensation
OH
OH
OH Si OH + OH Si OH
OH
OH
OCH3
OH
OH Si OH + H3CO Si OCH3
OCH3
OH
OH OH
OH Si O Si OH + H2O
OH OH
OH OH
OH Si O Si OH +(CH3OH)
OH OH
 Gelification, aging and drying
Pore size
si e
Channel communication
Time
Temperature
Catalyst
Morphology
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Optochemical sensors:
Main application fields
Process control
E i
Environmental
t l analysis
l i
Clinical ((bio)chemistry
)
y
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
STAGES OF INDICATORINDICATOR-BASED SENSOR
DEVELOPMENT
1 SELECTION OF THE COLORIMETRIC OR LUMINESCENT
1.
INDICADOR WHOSE OPTICAL PROPERTIES ARE MODIFIED IN
THE PRESENCE OF ANALYTE.
2. FABRICATION OF THE SENSITIVE PHASE (SUPPORT,
MEMBRANE, IMMOBILIZATION PROCEDURE).
DESIGN OF THE MOST APPROPRIATE SENSING SCHEME.
5. APLICATION AND VALIDATION OF THE
OPTODE TO THE TARGET ANALYSIS.

4. SPECTROSCOPIC AND ANALYTICAL
CHARACTERIZATION OF THE SENSOR.

3.



Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
3. Sensor arrays and exchangeable
h d Chemometrics.
heads.
Ch
t i
2. Composition
2
C
iti of
f
sensing membranes:
New polymer and antibiofouling materials.
4. Automatic serial
manufacturing Miniaturization,
manufacturing.
Miniaturization
NANOtechnology.
Lab-in-a-chip vs. chip-in-a-lab.
1. New
1
N
indicator
i di t d
dyes, (bi
(bio)chemical
) h i l
recognition elements. Molecularly
imprinted polymers. Novel transduction
mechanisms (photochemistry).
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Un BIOSENSOR es un dispositivo analítico que incorpora un
material biológico
g
((e.g.
g tejido,
j , microorganismo,
g
, enzima,,
anticuerpo, ácido nucleico, etc.) asociado a, o integrado en, un
transductor físicoquímico (óptico, electroquímico, térmico,
piezoeléctrico
i
lé t i o magnético).
éti )
BIOSELECTIVE
Membrane LAYER
(Biosens. Bioelectron. 2001, 16:121-131)
Exclusion/
protection
Signal
g
capture
and
processing
h

h
Data
OPTICAL
TRANSDUCER
ANALYTE
Interferents
SAMPLE
SELECTIVE
RECOGNITION
EVENT
ENERGY
TRANSDUCTION
SIGNAL
PROCESSING
READOUT
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Dependiendo de la naturaleza de la reacción bioquímica de
reconocimiento pueden clasificarse en:
A
B
A
• SENSORES BIOCATALÍTICOS
- También denominados sensores metabólicos. Basados en una
reacción catalizada
l d que origina un cambio
b en la
l naturaleza
l
química
de las especies involucradas (analito y otras).
Biológico: enzimas aisladas,
aisladas células,
células tejidos
(Biomimético: Polímeros de impronta molecular (MIPs))
• SENSORES DE BIOAFINIDAD
- Basados en interacciones específicas entre el analito y el bioreceptor.
No hay conversión del analito.
Biológico: anticuerpo, ácidos nucleicos (DNA/RNA),
receptores
(Bi i éti
(Biomimético:
P lí
Polímeros
i
impronta
t molecular
l l (MIPs),
(MIP ) oligopétidos,
li
étid oligonucleótidos,
li
l ótid
aptámeros)
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
PRINCIPIO DE MEDIDA
(A) El analito se convierte en un
PRODUCTO con propiedades
ÓPTICAS características (o
INDUCE un cambio en una señal
óptica: pH, desactivación fluor.,…).
(B) V
Variación
i ió de
d llas propiedades
i d d
ÓPTICAS de la ENZIMA en su
reacción con el analito
(C) El analito INHIBE una reacción
enzimática que produce/consume un
producto con propiedades ÓPTICAS
ANALITO
FENOL (O2)
ENZIMA
PPO
(Tirox.)
LACTATO
LMO
PESTICIDAS
AChE
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
O2
NH3
pH
CO2
Desactivación Oxidasa
luminiscencia
Glucosa,colina
colesterol
Lactato, fenol
Variación pH
Hidrolasa Urea
Variación pH
pH
Glucosa, urea
p
penicilinas
pesticidas
Variación pH Descarboxilasa Glutamato
TRANSDUCTOR
SUS
SUS
Enzima
Biocatalizador
PRO
PRO
Muestra
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
EJEMPLO: pesticidas
AChE
Acetilcolina + H2O
Ác. Acético + Colina
Transductor químico de
pH
Inhibidores de la AChe:
 Carbamatos
 Organofosforados
Indicador de tipo ftaleína
Cl
HO
OH
Intensa absorción en el visible
Elevada estabilidad fotoquímica
Cl
O
O
S
O
Inmovilización relativamente sencilla
a través de los grupos fenólicos
a través de los grupos fenólicos
pKa = 4.8 (amarillo) ‐ 6.4 (violeta)
ROJO DE CLORO
CLOROFENOL
ENOL
Biosens. Bioelectron. 2000, 14, 895
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
N
N
N
OCOCH3
2+
Ru
N
N
emmax = 610 nm
em = 0.02
= 0.58 s

Inhibidores de la AChe:
 Carbamatos
 Organofosforados
N
Inhibidor Substrato
H2O
AChE
N
N
N
2+
Ru
N
N
Buffer
OH
N
ENZIMA
D
emmax = 695 nm
em = 0.002
= 0.15 s
Orellana et al. Afinidad 2007, 64, 257–264
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
ANTICUERPOS
16 nm
Zona de unión del antígeno
Zona de
unión del
antigeno
Región
12 nm
variable
Cadena
ligera
Región
VH
CH1
Región
variable de la
cadena ligera
VL
Región
variable de la
cadena ligera
Región
constante de la
cadena ligera
CL
Fab
CH2
Región
constante de la
cadena pesada
Fc
constante
CH2
Cadena pesada
Heterodímeros con cuatro cadenas polipeptídicas: dos cadenas
pesadas idénticas y dos cadenas ligeras idénticas
Fuente: Dr. M.P. Marco (CSIC) Barcelona, Spain
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
ANTICUERPOS
Interacciones hidrofóbicas
Enlace de hidrógeno
Fuerzas de van der Waals
Interacciones electrostáticas
Anticuerpo antígeno
Anticuerpo-antígeno
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
ANTICUERPOS
Antígeno
marcado
Anticuerpo anti-analito
Transductor
Transductor
Respuesta
inversamente
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).
La propia
i molécula
lé l origina
i i
la
l señal
ñ l óptica.
ó i
Ensayo en fase heterogenea
Competitivo
No competitivo
Detección óptica
p
indirecta.
Se requiere un trazador o reacciones acopladas
para originar el efecto óptico.
Ensayo en fase heterogenea
Competitivo
p
No competitivo
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
H
H
N
O
H
S
O
CH 3
N
S
HO
CH 3
N
O
CH 3
O
H
N
CH 3
S
Cl
CH 3
N
COOH
N
CH 3
O
H
S
N
O
H
N
CH 3
CH 3
H
NH 2
H
S
N
CH 3
CH 3
O
COOH
Cloxacilina
O
H
CH 3
H
COOH
Oxacilina
H
N
N
O
CH 3
COOH
O
H
N
H
O
Cl
CH 3
O
O
N
N
CH 3
A
Amoxicilina
i ili
O
H
N
H
S
NH 2
Penicilina V
Penicilina G
H
H
COOH
COOH
Cl
H
CH 3
O
O
H
H
N
O
A
Ampicilina
i ili
O
H
S
N
O
Dicloxacilina
H
N
CH 3
CH 3
COOH
OC 2 H 5
H
H
S
N
O
Nafcilina
CH 3
CH 3
COOH
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
DERIVADOS DEL PIRENO
DERIVADOS DEL DANSILO
O
H H H
S
N
CH3
N
PAAPO
O
CH3
( )3
COOH
PBAP
N
H H H
S
N
NH
N
PAAM
O
CH3
O
O
N
CH3
COOH
DAP
COOH
CH3
CH3
COOH
O
O
H H H
S
N
( )3
NH
O
N
O
H H H
S
N
CH3
O
CH3
O
PBAM
COOH
H H H
S
N
NH
N
O
PAAX
S
NH
N
O
CH3
CH3
COOH
DAM
O
O
S
CH3
O
HO
N
CH3
O
O
O
H H H
S
N
H H H
S
N
N
CH3
CH3
COOH
G. Orellana et al. Patente ES 2197811; Anal. Chem. 2006
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Curva de calibrado
1.2
Ecuación de ajuste
A
 Amin
(B  B )
 max
 Amin
Señal normalizada = SN =
i
b
( B0  B )
 [analito ] 

1  
 IC 50 
Señal normallizada (SN)
1.0
0.8
0.6
0.4
02
0.2
0.0
0.000001
0.0
0.0001 0.001
0.01
0.1
1
10
100
[PENG], g mL
-1
Características Analíticas
IC
C50
30 ng mL-1
LD
2.4 ng mL-1
ID
(6.02 – 191) ng mL-1
r
0.998 (n = 5)
Moreno-Bondi, Orellana, et al., J. Agric. Food Chem. 2005, 53, 6635
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012
Catedrático de Universidad
Prof. Guillermo Orellana (UCM, Espanha) – Curso SENSORES ÓPTICOS QUÍMICOS – UEL/IAPAR – Londrina, PR – 22-23/032012