nolte ppt

MolecularInterferometry

TutorialMarch 28, 2007

Tutorial Outline

Introduction to Interferometry

Spinning-disc Interferometry (SDI)

The BioCD

Quadraspec, Inc.

Molecular Interferometric Imaging (MI2)

Interferometry

Quadrature and Interferometry

Im{E}

Re{E}Er

Es

Es

Re ETot{ }= Er + Es

I = Ir + Is + 2 IrIs cos rel + s t( )( )For rel = /2

I = Ir + Is + 2 IrIs sin s t( )( ) Ir + Is + 2 IrIss t( )

For Ir = Is

I = 2I0s t( )

2-Port Universal Response Curve

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Respon.qpc

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Phase Difference

/2 3/2 2

Esei (t )

Er

Er + Eseirel ei ( t )Port 1

Port 2

Signal-to-Noise Ratio (S/N)

Two choices: 1) Increase Signal: Resonance (difficult)2) Decrease Noise: High-speed Averaging (easy)

Operating Point (Spoke Height)

Quadrature

Operability Window (20%)

Quadrature

Q = 1

Resonance

Operating Point (Angle,Wavelength)

Operability Window (6%)

BiacoreSRU BiosystemsCorning Epic

Q = 10 to 100

Quadrature and Molecular ScatteringApparent Paradox:

Eloc

kEs = fElocei(kz-t)

Molecular scattering

Es is in-phase with Eloc

Im{E}

ElocEs

Amplitude

Kirchoff Integral:

Ed = i eikr

Re{E}

Elocd2x + fEloceikr

ei / 2

It is the continuous wave that is phase-shiftedupon diffraction, not the scattered.

Im{E}

Re{E}Efar

Es

Phase!

Interferometric Sensitivity (shot-noise limit):

Substrate

Focused Laser

Immobilized Biolayer

Direction of Disk Spin

Spoke.cd

NEM = 2hBPsQ

2

An 1( )vm

Noise-Equivalent Molecules:

N molecules detectedin area A.

Ps = 1 mWA = 50 m2vm = 5x10-19 cm3q = 0.7 = 500 nmB = 1 kHz

NEM 30 molecules

Spinning-DiscInterferometry

(SDI)

Focused Laser Beam

Ridge Height

Optical Load

QuadCD.cd

Quadrature

Land

Self-Referencing Interferometer

Far-Field Diffraction

Immobilized Antibody

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Gold SignalAb signalGold TheoryAb Theory

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Angle (Radians)

Land and Quadrature

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Land SignalGold SignalLand TheoryGold Theory

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Angle (Radians)

Varma, Nolte, et al. Biosens. and Bioelectron. 19 (11) pg. 1371-1376 (2004)

Why Spin?

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Frequency (Hz)

45 dB

Carrier frequency

DC

50 dB

50 dB Noise Suppression

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-9.10 10-3 -9.00 10-3 -8.90 10-3 -8.80 10-3antinode_tracescan15

DataHalf-harmonic

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Time (sec)

10-7

10-6

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10-4

10-3

0 100 2 104 4 104 6 104 8 104 1 105AvPower

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Frequency (Hz)

Full Disk Average

carrier

half-harmonicprotein

Differential Measurement

The BioCD

Antigen-Antibody Binding

Antibody-Assay (Label-free)

Antibody A Antibody B Antibody C Antibody A Antibody B Antibody C

Time

(incubation)

Sample with Analytes

Multi-analyte Label-free (mass sensing) High affinity (selectivity) Spatially addressed (RAM)

antigen A

antigen B

antigen C

nonspecific antigen

Exposing to Sample (incubation)

He-Ne 632.8 nm

Collimating lenses BS

Spinner

10x Objective BioCD

Image Plane

Fourier Plane

200 micron aperture

10 cm10 cm

Lock-in Amplifier

Detector

BioCD Far-Field Optics

Reversed Interferometric Response

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GoldAb+Ag

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Time (arb.)

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GoldAb+Ag

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Time (arb.)

/8 3/8

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Ridge Height ()Int.Respon.grp

Verify that signal is interferometric(not amplitude modulation) Move to opposite quadrant

Massive ChangeSi BioCD

1024 Au spokes onsilicon wafers

Synch pads

1024 spokes

QuickTime and aTIFF (LZW) decompressor

are needed to see this picture.







(Manoj Varma, 2002)

Fabrication-Free BioCD Classes

M. Zhao, et. al.Clin. Chem. 52, 2135(2006)

1) DifferentialPhase-Contrast

1. Cast a polyacrylamide gel stamp containing protein .

PDMS Spacer

SU8mold 2. Stamp the gel against the ODS

functionalized substrate.

Substrate

3. Remove the gel stamp

Polyacrylamide gel stamp with protein

Soft Lithography

1. Spin-coat photoresist over substrate functionalized with PSI/Biotin.

2. Expose and develop photoresist.

3. Soak in protein solution.

4. Remove photoresist.

Substrate

Photoresist

Photoresist Active surface

Protein solution

Immobilized protein

Photo-Lithography

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Time (sec)

rawscan.qpc

110 m 20 mProtein Ridgeson PC-class BioCD

DifferentialSignal

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Position (microns)

110 m

20 m

protscan.qpc

Disc Surface

Topology

Antibody ridges Soft lithography

Protein Patterning: Avidin on B-PSI w/ photolithographyRidge Height: 1 nmDetection: Phase Contrast at 50 kSamp/sec

2 mm

AB

A2 +B2

-2 nm

2 nm

-2 nm

2 nm

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2 nm

100 m

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(nm)

(mm) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 (mm)

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Protein Height Change (nm)

H/H

Photolith

Gel

H/M

Saturated Assay at 100 ng/ml

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Gel PrintPhotolith Print

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1-Specificity

Horse-AntiHorse

ROCGelLith.qpc

0.2% False Results

Receiver Operator Curve (ROC)

Silicon

SiO2

= / 2

ReferenceBiolayer

ReferenceSurface

/8

Fabrication-Free BioCD Classes2) In-Line Quadrature

Photonics West paper #6447-10

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Oxide Thickness (nm)

= 635 nm

Responsivity vs. Oxide Thickness

ResponOx.qpc

R = 0.018/nm

Antibody immobilization8 Head Piezoelectric Printer

Spot Uniformity: +/- 2%

CCD Head Camera Photo from Printer

Rendered Interferometric Data Through PicoMapsTM Software

Spotted Disk Layout:

Unit Cell

Ag A

Ag B

Spots:100 radial256 angular

= 25,600 spots= 6,400 unit cells

70 pl drops100 micron spots

Disc Scan:15,000 points per track @ 3.3 sec per point8x averaging per track1500 tracks @ 20 micron pitchBeam waist = 20 microns

Unit Cell

_ =

Post incubation

Target Reference

Pre incubation Difference

1 mm

Proteins are spotted into 2x2 unit cells of target and reference, providing good rejection of systematic shifts and non-specific binding to both spots.

Assay signal = dHtar/Htar dHref/Href

Data Analysis

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Root Height Variance (pm)

46 pm

Accuracy and Repeatability:

20 hours PBS-Casein Post-Pre scan h = 46 pm

Optics East Conf. (SPIE)Boston, MAOct. 3, 2006Paper 6380-20

Optics East Conf. (SPIE)Boston, MAOct. 3, 2006Paper 6380-20

46 pm46 pm

(nm)

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1

100 1000

Height Root Variance (pm)

BioCD

Surface PlasmonResonance

IntegratedWaveguides

3

0.3

0.0330 300

Sensit.qpc

BioCD Scaling Surface Sensitivity

Sm = min As = hm w02 = 0.2 pg/mm

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Concentration (ng/ml)

16% Biologically Active

KD = 35 ng/ml

QSSI-25020 Hour Incubation

Respon250.qpc

Assay Response Curve

Noise floor

Limit of Detection (LOD) scaling

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Number of Assays per Disc

2 ng/ml

100 assys

200 pg/mlk

D = 35 ng/ml

fact

= 17%

N = 0.1%k

D = 10 ng/ml

fact

= 50%

N = 0.01%

10,000assays

Performance of Silicon BioCDs(Jan. 25, 2007)

Property ValueLimit of Detection 100 pg/ml single assay

Scaling (expt. extrap.) 10 ng/ml at 1000 assaysSelectivity 10 ng/ml in 7 mg/mlHeight Resolution (@ 20 ) 20 pm

Under assay conditions 46 pmSensitivity (rand. noise) 0.2 fg/m

At 1 mm2 0.2 pg/mm2

West Lafayette, IN, at the Purdue Research Park incorporated Nov. 2004 3 rounds of investment funding currently at 40 employees delivered first product to vet reference lab March, 2007 manufacturing to fab 50,000 BioCDs per year @ $100per disc

Products & Manufacturing

BioCDsManufactured at Quadraspec, Inc.

(West Lafayette, IN)

Inspira SP250Manufactured at Medivative, Inc.

(Indianapolis, IN)

Inspira Lab StationManufactured at Medivative, Inc.

(Indianapolis, IN)

DiroChek Heartworm ELISA Serum Distribution

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Positive Serum SamplesNegative Serum Samples

QCHW Heartworm Assay Serum Distribution

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Positive Serum SamplesNegative Serum Samples

CHW Assay Dilution StudyCurrent Gold Standard Quadraspec

Detection at 1:35 Dilution Detection at 1:270 Dilution

MolecularInterferometric

Imaging(MI2)

Laser scanning vs. Full-field imaging

Laser

Objective

Detector

Laser scanning:Serial data acquisitionSpatial resolution (20m)Works dry.

120nm SiO2silicon wafer

CCD

120 nm SiO2silicon wafer

635nm filter

Objective

Full field imaging:Parallel data acquisitionHigh spatial resolution. (0.5m)Works dry and wet

Whitelight

SpinnerTranslation/Rotation stage

QSSI-1861 120nm 40x 635 nm filter

Microscope Image: 40x

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Shearing In-Line Interferometry

Image 1 Image 2 Difference

Diff = 2 I2 I1( )I2 + I1( )Shift Wafer

QSSI-1861 120nm 40x 300 400 500 600 700 800

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Scanning comparison

QSSI-903 120nm SiO2.

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QSSI-903 CV = 7%

Mean height = 1.3 nm = 0.2 ML

Ring height = 0.7 nm = 0.1 MLQSSI-1370 Dome height = 0.7 nm

QSSI-1709

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4x Objective

40x Objective

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Number of Averages

1N

1/f Noise

20 pm/pixel

repeat.fullfield.qpc

(0.5 m)

Full-Field

Scaling Comparison:

Laser Scanning:

hmin = 46 pm / pixel1 pixel = 20 m

S = 0.25 pg / mm

Full-Field:

hmin = 60 pm / pixel1 pixel = 0.5 m S = 0.01pg / mm

Scaling Comparison: Why so Good?

Laser Scanning:

Full-Field:fsamp = 106 / 0.1sec = 10MHz

fsamp = 150kHzN = 16

N = 1024

R = BWFullBWScan

= fsampFull NFull

fsampScanNScan

= 107 1024

105 16= 60

BioCD

Thank You

Question 1

Question 2

Question 3

Question 4

MolecularInterferometryTutorial OutlineInterferometryQuadrature and InterferometrySignal-to-Noise Ratio (S/N)Quadrature and Molecular ScatteringInterferometric Sensitivity (shot-noise limit)Spinning-DiscInterferometry(SDI)Historical Note: The Compact Disk (CD)Self-Referencing InterferometerFar-Field DiffractionWhy Spin?Differential Printing of ProteinDifferential MeasurementThe BioCDAntigen-Antibody BindingAntibody-Assay (Label-free)Exposing to Sample (incubation)BioCD Far-Field OpticsReversed Interferometric ResponseMassive ChangeFabrication-Free BioCD ClassesPhase Contrast DetectionSoft LithographyPhoto-LithographyProtein Ridgeson PC-class BioCDAvidin on B-PSI w/ photolithographyQuandrant DetectionSideband DemodulationDemodulation with AveragingSaturated Assay at 100 ng/mlReceiver Operator Curve (ROC)Fabrication-Free BioCD ClassesResponse CurveProtein SpotAntibody immobilizationSpotted Disk LayoutSpotted Disk LayoutData AnalysisAccuracy and RepeatabilityBioCD Scaling Surface SensitivityAssay Response CurveLimit of Detection (LOD) scalingPerformance of Silicon BioCDs West Lafayette, IN, at the Purdue Research Park incorporated Nov. 2004 3 rounds of investment funding currently at 40Products & ManufacturingCHW Assay Dilution StudyMolecularInterferometricImaging(MI2)Laser scanning vs. Full-field imagingMicroscope Image: 40xShearing In-Line InterferometryProtein Spot100 Micron Protein SpotScanning comparison Photo GalleryFull-FieldScaling ComparisonScaling Comparison: Why so Good?Fluctuations at 100 Molecules Per PixelScalabilityBioCDQuestion 1Question 2Question 3Question 4

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