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Lab-on-a-chip integration of silicon photonic p g pbiosensors for advanced point-of-care devices
Laura M. LechugaNanobiosensors and Bioanalytical Applications GroupReseach Centre on Nanoscience and Nanotechnology (CIN2) CSIC and CIBER-BBNBarcelona (Spain)Barcelona (Spain)
http://Nanob2a.cin2.es
DIAGNOSTICS: today and tomorrow
Limited to centralised laboratoriesRequired trained personnel
Clinical laboratory
Required trained personnelTime-consuming Expensive instrumentation
HIGH COSTSLOW SPEED OF ANALYSIS
Suitable for diagnostics in the field and in situBiosensor/POC devices
Suitable for diagnostics in the field and in-situFast, label-free, high sensitivityEnable permanent deployment and
unattended operationunattended operation
Biosensor technologies present attractive alternative to traditional laboratory diagnostics
Final Goal in diagnostics
biosensing evaluation treatmentsample
Science fiction reality in
X PRIZE Foundation and Qualcomm
ythe palm of your hand
Foundation Set to RevolutionizeHealthcare with Launch of $10 MillionQualcomm Tricorder X PRIZE
Th d i ill b t l bl f di i t f 15The device will be a tool capable of diagnosing a set of 15 diseases, with a total weight not more than 2 Kg
Biomedical & Clinical Diagnosis requirements
Biomedical & Clinical Diagnosis requirements
• Sensitive and selective detection in clinical Optical Biosensors
matricesAble to detect relevant levels of target molecules in body fluids
Label-free assays
Real time measurements• Label-free assays
• Real time measurements
High sensitivity
Multiplexing
• Direct detection from real samples
• Multiplexing capability
Direct Detection
p g p ySimultaneously analyse from 10 to thousands of biomolecular interactions
• Easy manipulationEasy manipulationFast time-to-result, assay simplicity, low volume of samples and reagents
• Manufacturability• ManufacturabilityCost effective and scalable for mass production
Photonic Biosensors
EvanescentEvanescent Wave Wave DetectionDetectionBinding event,
Effective refractive index modification: changes in the optical path
AnalyteAnalyte
RI ~ (dn/dc)vol EW: 100-900 nm
Change of refractive index, n
Change of optical parameter
p ~
Change of optical parameter, p
(Intensity,Phase, polarisation, resonant momentum ) Hi h iti it i ll t th fresonant momentum,…) High sensitivity, specially at the surface
Direct measurement without labelling of molecules (LABEL-FREE)
Real time (binding can be continuously monitoring) Small volumesReal-time (binding can be continuously monitoring). Small volumes
Detection of selective biomolecular interactions. Generic technology
Why integrated optical biosensors?
High sensitivity; integration of sources, sensors, detectors, flow system,electronics and data processing on a compact platform; Si and polymer technologymass production: Low cost fabrication Multiplexing: Sensor packed arrays
p
mass production: Low-cost fabrication. Multiplexing: Sensor packed arrays
nsor
chi
p
Device physics and engineering: modeling, fabrication and characterization
Bio
sen Integration of components:
microfluidics, light coupling, photodetectors
Surface biofunctionalization and full
e m
Surface biofunctionalization and full development of applications
Port
able
Plat
form
Electronics, software, hardware, wireless
BIOSENSORS Li it f d t ti B lk S iti it
Sensitivity comparision
BIOSENSORS Limit of detection (pg/mm2)
Bulk Sensitivity
SPR (LSPR) 0.5-1 10-5-10-7
NSO
RS
Grating Couplers 0.3 2.10-6
Integrated Mach Zehnder 0 01 0 06IC B
IOSE
Integrated Mach-Zehnder Interferometer
0.01-0.06 1·10-7-2·10-8
Young interferometer 0.013-0.75 9·10-8-9·10-9
PHO
TON
I
Microring resonators 3-15 5.10-5-7·10-7
Photonic Crystals 0 4 7 5 ~10-5HE-
AR
T: P
Photonic Crystals 0.4-7.5 ~10 5
Silicon wires 0.25 2. 10-6
ATE-
OF-
TH
Slot waveguides 0.9-16 ~10-6
STA
R l t iti iti f M fM l b l f d t ti
eff
eff
o N
dN
Cddn
nn
Calculated accordingRelevant sensitivities for pM-fM label-free detection
Laser & Photonics Reviews 6, 463-487 (2012)2012 Breakthroughs. IEEE Photonics Journal (2013) In press
THE LAB ON CHIP PROJECTTHE LAB-ON-CHIP PROJECT
“Lab-on-a-chip” Biosensor platform
Surface biofunctionalization
Integrated MicroFluidics Sample extraction
Optical readout (PD)
Grating couplers
Array of interferometric sensors
Signal modulation(all optical modulation principle)
ElectronicsCMOS chip
The integration of different components into a chip is a challenge. The integration is not trivial due to destructive integration processes and performance optimality.
THE SENSOR CHIPTHE SENSOR CHIP
Mach-Zehnder Interferometer biosensor
2 cos( ( ))
2 2 ( )
T S R S R S
S eff S R
I I I I I tL LN N N
E t
S eff S R
• Single mode behaviour Evanescent
wave detection • High surface sensitivity
• Operating in visible range
F b i t d ith t d d• Fabricated with standard microelectronics technology
a Si3N4 (nc=2.00) core layer, 250 nm thick (TM);75 nm (TE)
Single mode rib waveguides
Width 4 m; rib depth: 1- 4 nm a SiO2 cladding layer, 2 m (n=1.46)
Si3N4 75 nmWidth 4 m
ribrib ofof 4 nm4 nm
Si3N4 75 nmWidth 4 m
ribrib ofof 4 nm4 nm
Si3N4 75 nmWidth 4 m
ribrib ofof 4 nm4 nm
Si Sustrate
Si3N4 75 nm
SiO2 2 m claddingSi Sustrate
Si3N4 75 nm
SiO2 2 m cladding
Si3N4 75 nm
SiO2 2 m cladding
= 600-700 nm
Interferometric biosensors
MACH-ZEHNDER INTERFEROMETER (MZI) BIMODAL WAVEGUIDES (BiMW)
Rib ≤ 4 nmRib ≤ 4 nm
WO/2009/010624,PCT/ES08/070142 12/669307 (US)
Bimodal waveguide interferometer
He‐Ne Laser
Microfluidic header Photodetector
objectiveTemperaturecontroller
Objective
ChipCopper base• Total length is 30 mm (bimodal 25 mm)
chipAdquisition
card
Sensitivity evaluation
Objective• Sensing window:15 mm • 16 independent sensors
Two‐Sections PD
Sensitivity evaluation
no min= 2.5 x10-7
Direct detection in the picomolar range (10-12 M)
no,min 2.5 x10• One of the most sensitive EW sensors• a simple IO sensor • visible rangeg ( )
(fg/mm2) • visible range• hundreds of devices per wafer
Hi h iti itHigh sensitivityMicrosystems platformsMultiplexing capabilities
J. Lightwave Technology 29, 2011, 1926-1930
THE MICROFLUIDICSTHE MICROFLUIDICS
µ-Fluidics integration
Hermetic sealingNo air bubblesNo air bubblesLow cost (disposable)Affording multiplexing
Two technologiesPDMS and/or PMMA: independent flow cells, reusableFull integration with SU-8 at the wafer level
Bonding (300 kPa, 100ºC)
Fluidic channel (15 µl) tubingAssembly
g ( )
PMMA master PDMS mold4 independent channels
volume: 15 µL
Integration sensor/SU-8 microfluidics
• SU-8 based microfluidic to obtain a 3D platform with a dedicated microchannel on top of each sensor.• Connections with standard fluidic connectors and O-i i f t li
MZI
56µm20 µm height
rings, ensuring perfect sealing• Standard polymer processing at wafer level:
Channels of 20-100 µmOnly µ-Fluidics
56 µm height
Channels of 20-100 µm height and 50-150 µm width
µ-Fluidics & chip
BIOFUNCTIONALIZATION & BIOSENSING
Biofunctionalization of the sensor area
Cl iCOOH
Si
COO-Na+SILANIZATION OF THE Si3N4 SURFACEi) Oxidation (O3, 1h)ii) Activation (HNO3, 25 min, 75ºC)iii) CTES Cleaning
Si3N4
OH
SiOH
O-Na+OH
i) Ac/EtOH/H2O
ii) SDS 1%iii) HCl:MeOH (1:1) Hydrogen
bonding
SiOH
OOH
OH OOH
H H
iii) CTES iv) 120ºC, 1h
OH OHOH
Oxidation
bonding OH O
i) UV/O3 1h
NHN
R’
Bondformation
ii) 10% HNO3 75°C 25 min, reflux
BIOFUNCTIONALIZATION
COOH
[ 1h, 110 °C]
CO O
NR
NCNR
R’EDC/NHS 10 minC
O NHNH2
O
SiOOO
SiOO
EDC/NHS 10 min(0.2/0.05 M)
ActivationPeptidic bondO
Si
C
OO
JCIS, 2012, In press
Diagnostic applicationDetection of Human Growth Hormone (hGH)
BiMW0,6
0,7Essential for normal growth and development. Regulates metabolism throughout all adult life.
BiMWLOD: 8 pg/mL, 160 fMIC50: 35 ng/mL
0,3
0,4
0,5
(r
ad) Inhibition
immunoassay
IC50: 35 ng/mLLinear range: 680 pg/mL- 4 µg/ml
0,0
0,1
0,2
SPR
-10 -8 -6 -4 -2 0 2-0,1
log (µg/mL hGH)
SPRLOD: 4 ng/mL, 200 pMIC50: 91 ng/mL
SPR
Linear range: 18-542 ng/ml
The total assa time has been red ced to 13 minThe total assay time has been reduced to 13 minThe LOD has been improved 1000 timesThe linear range has been widely increased
Direct Immunosensing detection of pathogens with BiMW
• Tested with PAb for Pseudomonas Aureginosa (Pseu. A.) (Anti Pseu: ab68538)• Thermally inactivated (antigens and bacteria)• Control with S. Aureus (Staphylococcus aureus)
0 25
0,30
( p y )
0,20
0,25 Direct immunoassay
0,15
ra
d
LOD 10 cfu/mL
0 05
0,10x
LOD 10 cfu/mL
100 101 102 103 104 105 106 107 1080,00
0,05
10 10 10 10 10 10 10 10 10
cfu/mLWith SPR, 100 cfu/mL
THE GRATING COUPLER
Light coupling by Grating Couplers
Direct Focusing Diffraction Grating Coupler
θ Towardsθ
ΛSi3N4
Towardsinterferometers
Only in lab conditionsGratings provide higherstability of light coupling.
3 4
SiO2
Period: 300-500 nm
Fabrication by Electron Beam Lithography
stability of light coupling. Period: 300 500 nmDepth: 20-40 nm
Λ = 400 nm on a 20 µm width taperSEM pictures of a grating (Λ = 400 nm, DC = 0.5, depth = 40 nm)
Light coupling by Grating Couplers
Fiber pigtailed laser diode laser λ=658 nm, TE polarization
Optical characterization, p
450 nm‐period grating, θ=10°30’
Better integration and stability than end-fire method. Good alignment tolerance: angle tolerance Good alignment tolerance: angle tolerance of ≈1° at -3dB and position tolerance of ≈ 50 µm at -3dB
BiMW with Grating Couplers: sensing performance
• Changes Δφ induced by refractive index variations (HCl, from 0.05 to 0.5 M)• BiMW excited via a grating coupler (period: 450nm, depth: 40 nm, DC 0.5)
I = 65 0 mA
10
Experimental data Linerar fit20 20
42 43 44 45
Ilaser = 65,0 mAΘ = 9°35’
6
8ra
d)
0 0
20
%)
4 (2
r
40
-20
S (%
)
40
-20S (%
1 10-3 2 10-3 3 10-3 4 10-3 5 10-30
2
106 108 110 112
-60
-40
42 43 44 45
-60
-40
Limit of detection: 3.3·10-7 RIU
(a) Sensor response to the injection of HCl 0.05 M in the sensing area, (b) calibration curve d ( ) t th i j ti f HCl 0 4 M i th i
1x10 3 2x10 3 3x10 3 4x10 3 5x10 3
n106 108 110 112
Time (min)42 43 44 45
Time (min)
Δφ versus Δn and (c) sensor response to the injection of HCl 0.4 M in the sensing area
IEEE Photonics Journal, 2013, In press
FINAL INTEGRATION
Lab-on-chip integration
Surface biofunctionalization
Sample extraction
Integrated polymer microfluidics
Optical readout (PD)
Integrated MicroFluidics Sample extraction
Array of sensors
Grating couplers
f f
Signal modulation(all optical modulation principle)
Array of interferometric sensors
ElectronicsCMOS chipGrating couplers
θ
ΛSi3N4
SiO2
Towardsinterferometers All-optical signal modulation
25
30
10
15
I2 I3
Under technological transfer analysis
1056 1058 1060 1062 1064 1066 1068 1070
0
5
10
15
20
S
(rad)
time (min)1056 1058 1060 1062 1064 1066
-10
-5
0
5
I2, I
3 (a
.u.)
•EP2017602; PCT/ES08/070142; CA2693423, CN101842691, US20100271634 (Granted 2012), JP2010533849
• EP2278365 (Granted 2012), PCTES08070142, CA2693423, CN102077124, US20110102777 (Granted 2012), JP2011519071
time (min)time (min)
Lab chip Lab on chip 12, 1987-1994 (2012)
AcknowledgementsN bi d Bi l ti l A li ti G (CIN2 CSIC)Nanobiosensors and Bioanalytical Applications Group (CIN2-CSIC)
and Chemical Transducer Group (IMB-CNM-CSIC)Team profile: physicist, chemist, electronics engineers, molecular biologist,biotechnologist, biophysics, optoelectronics technology,…..
Finantial funding from:European Union MINECOCIBER-BBNM. Botín Foundation
SpinSpin offs:offs:SpinSpin--offs:offs:SENSIA, SLSENSIA, SLBIOD, SLBIOD, SL
Nanobiosensors and Bioanalytical Applications Group
From fundamental physics to the implementation of diagnostic nanodevices for real applications
(Nano)Plasmonics: SPR, Magneto-SPR and LSPR Integrated optics: Nanophotonic biosensor (MZI)Nanomechanical biosensors (standard and optical)Biofuncionalization with biological receptorsMicrofluidics integrationLab-on-a-chip platforms (in-vitro and in-vivo)
MORE INFO
Applications: clinical diagnostics, environmental control
MORE INFO nanob2a.cin2.es