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)

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Applications: clinical diagnostics, environmental control

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