biosensors group...a nanomechanical motion (snps-dna and microcantilever femtomolar-proteins) 2560...
TRANSCRIPT
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Dr. Laura M. LechugaInstituto de Microelectrónica de MadridCentro Nacional de Microelectrónica, CSIC
Biosensors GroupBiosensors GroupNanobiosensorsNanobiosensors and molecular and molecular nanobiophysicsnanobiophysics GroupGroup
(Centro de Investigación en Nanociencia y Nanotecnología, ICN-CSIC)
nanoMZI
SU8 microchannel
Silicon Nitride (wg
core layer)Silicon Oxide (wg
cladding layer)
SU8 microchannel
Silicon Nitride (wg
core layer)Silicon Oxide (wg
cladding layer)Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
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OH
Si
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OH
Si
N
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OH
Si
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OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
nanoMZI
SU8 microchannel
Silicon Nitride (wg
core layer)Silicon Oxide (wg
cladding layer)
SU8 microchannel
Silicon Nitride (wg
core layer)Silicon Oxide (wg
cladding layer)Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
Si
N
O
OH
SPR
Micro-MZI
MZI
Microfluidics
OpticalMicrocantilevers
Nanomechanics
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Enabling technologiesEnabling technologies– Transducers (Plasmonics, magnetoplasmonics, integrated optics,
nanomechanics, carbon nanotubes)– Bio-Surface chemistry (covalent and affinity binding)– Design, materials, technology and packaging– Optical and magnetic tweezers set-up for fundamental studies
CNM BiosensorsCNM Biosensors– Portable Surface Plasmon Resonance Biosensor (commercial
device)– Magnetooptical Surface Plasmon Resonance Sensor– Integrated Mach-Zehnder Photonic Interferometer nanosensors
(Si, polymer technology)– Nanomechanical biosensors (Si technology). Optical waveguide
cantilevers– Carbon nanotubes biosensors
OurOur ActivityActivity in in BiosensorsBiosensorsDevelopment of portable multiBiosensor Prototypes, mainly fabricated with Microelectronics (MEMS) Technology, at micro- and nanoscale, for lab-on-a-chip Microsystems applicationapplication in human health diagnostics, environmental control, defense, genomics and proteomics.
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PLASMONICSPLASMONICS
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Product: Plasmonic Biosensor (SPR)
bombaperistáltica
bombaperistáltica
MUESTRA
AGUA
bobinade mezcla
válvulainyección
válvula6 vías
LASER(luz colimada)
REFLECTOMETRÍA
Lámina ORO
Lente
bombaperistáltica
bombaperistáltica
MUESTRA
AGUA
bobinade mezcla
válvulainyección
válvula6 vías
LASER(luz colimada)
REFLECTOMETRÍA
Lámina ORO
Lente
Flow delivery SystemWireless
transmission module
Electronics
FLOW CELLV=300 νl
flow rate = 1-100 μl/min
θ
θPRISM
LASERλ = 670 nm
Si-PHOTODETECTOR
GOLD FILMd = 45 nm
BEAMSPLITTER
5 mm
FLOW CELLV=300 νl
flow rate = 1-100 μl/min
θ
θPRISM
LASERλ = 670 nm
Si-PHOTODETECTOR
GOLD FILMd = 45 nm
BEAMSPLITTER
5 mm
θ
θPRISM
LASERλ = 670 nm
Si-PHOTODETECTOR
GOLD FILMd = 45 nm
BEAMSPLITTER
5 mm
θ
θPRISM
LASERλ = 670 nm
Si-PHOTODETECTOR
GOLD FILMd = 45 nm
BEAMSPLITTER
5 mm
Sensor module
N
O
S
H
Au
N
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
HN
O
S
H
Immobilizationand regeneration
chemistry
Software
www.sensia.es
Au 45 nmSPR working principle
PlasmonicsPlasmonics
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SPR Sensor: SPR Sensor: Detection of environmental pollutantsDetection of environmental pollutants
www.sensia.es
1E-6 1E-5 1E-4 1E-3 0,01 0,1 1 10 100 10000
20
40
60
80
100
SP
R S
IGN
AL
Analyte concentration (μg L-1)
DDT Chlorpyrifos Carbaryl
Detection limit:0.02-0.05 μg/l
EU Drinking waterquality directive98/83 (maximumlevel of pesticidesin water is 0.1 μg/l)
1E-4 1E-3 0,01 0,1 1 10 100 1000 10000
0
20
40
60
80
100
120 MiliQ Water Drinking Water River Water Groundwater
SPR
SIG
NAL
Chlorpyrifos (μg L-1)
Real sample analysis
Determination of main environmental pollutants at ppt levels
Direct detection in real samples without pretreatment
Reduced amount of sample and reagents
complete cycle of analysis in 20 min.
Simultaneous determination up to 6 analytes
PlasmonicsPlasmonicsIn collaboration with Ci2B-UPV
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SPR Sensor: Clinical diagnosisSPR Sensor: Clinical diagnosis
1E-6 1E-5 1E-4 1E-3 0,01 0,1 1 10 100-10
0
10
20
30
40
50
60
70
80
90
100
110
hGH µg/mL
Human serumUrinePBST
1E-6 1E-5 1E-4 1E-3 0,01 0,1 1 10 100-10
0
10
20
30
40
50
60
70
80
90
100
110
1E-6 1E-5 1E-4 1E-3 0,01 0,1 1 10 100-10
0
10
20
30
40
50
60
70
80
90
100
110
hGH µg/mL
Human serum
PBST
SP
R S
IGN
AL
Detection limit:6 ng/ml
• Detection limit: 6 ng/mL – 300 pM
• I50: 140 ng/mL – 7 nM
• Working Range: 20 – 1120 ng/mL1 – 56 nM
• Cycle time: 25 min / 2 samples
• Number of cycles: 100 cycles / cell
Determination of a clinical analyte at physiological levels
Detection limit < 1 nM
Direct detection in human serum and urine without pre-treatment
Reduced amount of sample and reagents
Analysis of the Human Growth hormone
PlasmonicsPlasmonics
1 2 3 4 5 6 7 8 9ELISA
SPR
0
5
10
15
20
25
30
35
40
hGH ng/mL
Sample
Growth hormone concentration in human samples (serum)
ELISASPR
In collaboration with DIO-CNB-CSIC
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LOD = 10nM
0,01 0,1 10
2
Ref
ract
ive
inde
x (a
.u)
Concentration (μM)
Hybridization of 25 mer complementary target to a breast cancer gene
25T15mer 0,5 mM SAM
0 500 1000 1500 2000 2500
46,0
46,5
47,0
47,5
48,0
48,5
49,0
Cha
nge
on re
fract
ive
inde
x
Time (s)
Complementary 5μM Control 5μM
Hybridisation detection
Secuence with a SNPs
SNPNucleotides
Normal Secuence
0 200 400 600 800 1000 1200 1400-0,2
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
Indi
ce d
e re
fracc
ion
(u.a
)
ΔV= 1,16
ΔV= 0,96ΔV= 0,89
Complementario Mismatch externo Mismatch interno Secuencia control
Tiempo (s)
SNPs detection
Early detection of hormone cancer through specificProteins Biomarkers
SPR Sensor: Clinical diagnosisSPR Sensor: Clinical diagnosisEARLY DETECTION OF INHERITED BREAST CANCER
Detection of point mutations at BRCA-1
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MAGNETOPLASMONICSMAGNETOPLASMONICS
In collaboration with Magnetic Group IMM-CNM
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MagnetoMagneto--Plasmonic BiosensorPlasmonic Biosensor
Sensitivity up to one order of Sensitivity up to one order of magnitude higher that the SPRmagnitude higher that the SPR
0 100 200 300 400 500
0
10
20
Time (s)S/
S min
MOSPR SPR
1. Combination of SPR and magneto-optic activity Great enhancement of the magneto-optic effects
2. Magneto-optic effects strongly depend on the refractive index of the dielectric
Biosensing application
θ
θ
Glass prism
He-Ne Laser
Photodiode
Magneto-optic multilayer
Rotating magnetization
Flow cell
Rotating magnets
Mρ
)(tRR
pp
ppΔ
θ
θ
Glass prism
He-Ne Laser
Photodiode
Magneto-optic multilayer
Rotating magnetization
Flow cell
Rotating magnets
Mρ
)(tRR
pp
ppΔ)(tR
Rpp
ppΔ)(tR
Rpp
ppΔ
PlasmonicsPlasmonics
ΔnEmin
MOSPR 5·10-6
SPR 1.5·10-5
Signal improvedthree times
(PCT/EP2005/006273, licensed to Sensia,SL)
External medium
Receptor
Analyte
31 nm Gold layer
εdεd
SPxk
12 nm Cobalt layer
External medium
Receptor
Analyte
31 nm Gold layer
εdεd
SPxk
12 nm Cobalt layer
Ferromagnetic layer
Metal layer
In progress: nanostructured ferrromagnetic layers: HIGHER SENSITIVTY
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PHOTONIC BIOSENSORSPHOTONIC BIOSENSORSIn collaboration with Transducer Group IMB-CNM
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Integrated Photonic Micro/Integrated Photonic Micro/nanobiosensorsnanobiosensors
IntegratedIntegrated OpticsOptics
Δno,min= 1.5x10-7 (TM)ΔNeff,min= 2.5x10-8 (TM)
0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.0070
5
10
15
20
25
30
Phas
e ch
ange
ΔΦ
Refractive index variation Δn
TE exp TE theorical y=4472.004X+0.948
Sensitivity evaluation
The detection limit value corresponds to an average growth layer of 1.10-4 nm (3.105 mol.cm2) and a Surface Sensitivity around 2.10-4 nm-1. Direct detection in the picomolar range (10-12 M) is possible (60 fg/ mm2)
Si Sustrate
Si3N4 75 nm Width 4 μm
ribrib ofof 4 nm4 nm
SiO2 2 μm claddingSi Sustrate
Si3N4 75 nm Width 4 μm
ribrib ofof 4 nm4 nm
SiO2 2 μm cladding
Si3N4 75 nm Width 4 μm
ribrib ofof 4 nm4 nm
SiO2 2 μm cladding
50 μm50 μm50 μm
4 nm
MZIMZI
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DNA targetDNA probe
Sensor area
Reference areaLight input
DNA targetDNA probeDNA probe
Sensor area
Reference areaLight inputLight input
1E-12 1E-11 1E-10 1E-9 1E-8 1E-7 1E-6
0,0
0,2
0,4
0,6
0,8
1,0
Data: Data13_BModel: LangmuirEXT1 Equation: y = (a*b*x (1-c))/(1 + b*x (1-c)) Weighting:y No weighting Chi 2/DoF = 0.00597R^2 = 0.96656 a 0.86261 ±0.08076b 125409798.76584 ±6350.78103c -0.0351±0.27267
Sign
al (A
. U.)
DNA (M)
By SPR only 100 nM is possible!!!
control
SNP, LOD = 10 nM
[ADN] (M) 58 MER1E-12 1E-11 1E-10 1E-9 1E-8 1E-7 1E-6 1E-5 1E-4
0,0
0,5
1,0
1,5
2,0
2,5
3,0
Δφ (2
π) LOD = 10 pM
Control Complementary
By SPR only 10 nM is possible!!!
Photonic MZI Sensor: Clinical diagnosisPhotonic MZI Sensor: Clinical diagnosisEARLY DETECTION OF INHERITED BREAST CANCER
Detection of point mutations at gene BRCA-1
Secuence with a SNPs
SNPNucleotides
Normal Secuence
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Fibre In
GRIN layerGRIN layer
Input coupling Air
AR film
Si
SiO2
Air
AR film
Si
SiO2
Optical readout
MicroFluidics and surface functionalization
Sensor design
II
Signal modulation
Integrated Photonic Integrated Photonic MicrosystemMicrosystem
Sensor MZI
Colaboración con IKERLAN
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NANOMECHANICS BIOSENSORSNANOMECHANICS BIOSENSORSStandard and optical microcantilevers
In collaboration with Transducer Group IMB-CNM
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Nanomechanical Nanomechanical BiosensorsBiosensors
NanomechanicsNanomechanics
A new class of highly sensitive, label-free and direct biosensor which transduces the molecular recognition of biomolecules into a nanomechanical motion (SNPs-DNA and femtomolar-proteins)microcantilever
2560 cantilevers, 0.334 μm thick cantilevers, k= 5.5.10-3
Six time more sensitive for biosensing than commercial ones
0 50 100 150 200
-100
-50
0
50
LiB-DDT 5μg/ml
HCl 0.1M
HCl 0.1M
DDT 10nMLiB-DDT 5μg/mlLiB-DDT 5μg/ml
Tiempo (min)
Def
lexi
ón (n
m)
Antibody AntibodyAntibody
+
90 100 110 120 130
-1200
-800
-400
0
Time (min)
Def
lect
ion
(nm
)
PROTEIN EVALUATION(nM range)
DNA EVALUATION
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Integration in MicrosystemsIntegration in Microsystems
•Optics:20-VSCEL+Drivers+ Photodetector array. CMOS circuitry
•Microarrays of 20 microcantilevers
•Microarray/Microfluidics integrated
•Integrated and portable device
www.optonanogen.com
IST-2001-37239
For measuring arrays of microcantilevers......
NanomechanicsNanomechanics
Novel optical waveguide microcantilever sensorNovel optical waveguide microcantilever sensor
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1997 2000 2001
Starting Starting Biosensors Biosensors
GroupGroup
Starting work in Starting work in nanobiosensorsnanobiosensors SENSIA, SLSENSIA, SL
EvolutionEvolution
ProductsProductsPortfolioPortfolio
PersonnelPersonnel 2 85 16
Operative Operative biosensorsbiosensors
Research LinesSPR Biosensor
Integrated OpticsBiosensors
Research LinesSPR Biosensor
Micro interferometer
Research LinesSPR Biosensor
Nanophotonics interferometerNanomechanical biosensors
2004
50 μm50 μm50 μm
Biosensors Group: MAIN ASSETSBiosensors Group: MAIN ASSETS
Product LinesSPR Biosensor
Magnetoplamonics sensorNanophotonics interferometerNanomechanical biosensors
Specific applicationsMicrofluidics