Molecular Interferometry

Tutorial March 28, 2007

Tutorial Outline
Introduction to Interferometry Spinning-disc Interferometry (SDI) The BioCD Quadraspec, Inc. Molecular Interferometric Imaging (MI2)

Interferometry

Quadrature and Interferometry

Port 1

2-Port Universal Response Curve

1

0.8

E se

i ( t )

E r + E se i rel e i ( t )
Port 2

Intensity

0.6

0.4

0.2

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

Respon.qpc

/2

Phase Difference

3 /2

Im{E} Es Es Re{E} Er

I = Ir + Is + 2 Ir Is sin( s (t ))
Ir + Is + 2 Ir Is s (t )
For Ir = Is

I = 2 I0 s (t )

Re{E Tot } = E r + E s

Signal-to-Noise Ratio (S/N)

Two choices: 1) Increase Signal: Resonance (difficult) 2) Decrease Noise: High-speed Averaging (easy)
Biacore SRU Biosystems Corning Epic

Quadrature
Operability Window (20%)

Resonance
Operability Window (6%)

Q=1 Q = 10 to 100
Quadrature

Operating Point (Spoke Height)

Operating Point (Angle,Wavelength)

Quadrature and Molecular Scattering

Apparent Paradox:

Im{E}

Molecular scattering Eloc k Es = fElocei(kz-t) Es is in-phase with Eloc

Eloc

Es

Re{E}

Amplitude

Kirchoff Integral:

Im{E} Es

E d = i e ik r E loc d 2 x + fE loce ik r

Efar

Phase! Re{E}

e i / 2
It is the continuous wave that is phase-shifted upon diffraction, not the scattered.

Interferometric Sensitivity (shot-noise limit):

Noise-Equivalent Molecules:
Focused Laser Immobilized Biolayer

NEM =

2hB A PsQ 2 (n 1)v m

Substrate Direction of Disk Spin

Spoke.cd

N molecules detected in area A.

Ps = 1 mW A = 50 m2 vm = 5x10-19 cm3 q = 0.7 = 500 nm B = 1 kHz

NEM 30 molecules

Spinning-Disc Interferometry (SDI)

Self-Referencing Interferometer
Focused Laser Beam

Quadrature

Optical Load Ridge Height

Land

QuadCD.cd

Far-Field Diffraction

Land and Quadrature

1.0
Land Signal Gold Signal Land Theory Gold Theory

Immobilized Antibody
0.600
Gold Signal Ab signal Gold Theory Ab Theory

0.8

0.500

Normalized Signal

Difference Signal
-0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08
Iexpt.grp

0.400

0.6

0.300

0.4

0.200

0.2

0.100

0.0 -0.08

0.000 -0.08

-0.06

-0.04

-0.02

0.02

0.04

0.06

0.08
DiffIexpt.grp

Angle (Radians)

Angle (Radians)

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

Why Spin?
100.0 80.0 60.0 dB 40.0 20.0 0.0 -20.0 -5.00 104

50 dB Noise Suppression
DC Carrier frequency

50 dB
45 dB

Power.out

0.00 100

5.00 104

1.00 105

1.50 105

2.00 105

Frequency (Hz)

0.20

Differential Measurement
0.18 Volts 0.16

Data Half-harmonic

0.14

0.12
antinode_tracescan15

10-3

carrier

-9.10 10-3

-9.00 10-3 Time (sec)

-8.90 10-3

-8.80 10-3

10-4 Power half-harmonic protein 10-5

10-6

10-7
Full Disk Average
AvPower

0 10

2 10

4 10

6 10

8 10

1 105

Frequency (Hz)

The BioCD

Antigen-Antibody Binding

Antibody-Assay (Label-free)
Sample with Analytes

Time (incubation)

Antibody A

Antibody B

Antibody C

Antibody A

Antibody B

Antibody C

antigen A antigen B antigen C nonspecific antigen

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

Exposing to Sample (incubation)

BioCD Far-Field Optics

He-Ne 632.8 nm

10x Objective Collimating lenses BS

BioCD

Spinner Detector 10 cm 10 cm

Lock-in Amplifier

Fourier Plane 200 micron aperture

Image Plane

1.00

Interferometric Intensity

0.80

Reversed Interferometric Response

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

0.60

0.40

0.20

0.00

Int.Respon.grp

0.1

0.2

0.3

0.4

0.5

Ridge Height ()

/8
1.20 Gold Ab+Ag 1.00 Normalised Signal
Normalised Signal 1.20 1.00

3/8
Gold Ab+Ag

0.800

0.800

0.600

0.600

0.400 -200 -100 0 Time (arb.) 100 200

0.400 -200 -100 0 Time (arb.) 100 200

Si BioCD
1024 Au spokes on silicon wafers

Massive Change

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Synch pads
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1024 spokes

(Manoj Varma, 2002)

Fabrication-Free BioCD Classes

1) Differential Phase-Contrast

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

Polyacrylamide gel stamp with protein

Soft Lithography

Substrate SU8 mold

2. Stamp the gel against the ODS functionalized substrate.

PDMS Spacer

3. Remove the gel stamp

1. Cast a polyacrylamide gel stamp containing protein .

Photo-Lithography
Protein solution Photoresist Substrate

1.

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

Active surface

3. Soak in protein solution.

Photoresist

Immobilized protein

2. Expose and develop photoresist.

4. Remove photoresist.

0.04

Differential Intensity (I/I)

Protein Ridges on PC-class BioCD

110 m

20 m

0.03 0.02 0.01 0 -0.01 -0.02 -0.03 -0.04 0 10 20 30 Time (sec) 40

rawscan.qpc

Differential Signal

Antibody ridges Soft lithography

50

10

110 m 20 m

8 Protein Height (nm)

Topology

0 0

Disc Surface
protscan.qpc

200

400

600

800

1000

Position (microns)

Protein Patterning: Avidin on B-PSI w/ photolithography Ridge Height: 1 nm Detection: Phase Contrast at 50 kSamp/sec

2 mm

2 nm

A
A 2 +B2

2 nm

-2 nm

2 nm

B
0 nm
100 m

-2 nm

(nm) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 (mm)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 (mm)

(nm) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 (mm)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 (mm)

Saturated Assay at 100 ng/ml

0.08 H/M 0.06 Probability Gel 0.04 H/H Photolith

0.02

0 -1 -0.5 0 0.5 1 1.5 2 Protein Height Change (nm)

Receiver Operator Curve (ROC)

0.2% False Results
1

0.99 Sensitivity

0.98

0.97

Horse-AntiHorse

Gel Print Photolith Print 0.96 -0.01

ROCGelLith.qpc

0.01

0.02

0.03

0.04

0.05

0.06

1-Specificity

Fabrication-Free BioCD Classes

2) In-Line Quadrature

= / 2
SiO2 Silicon

/8
Photonics West paper #6447-10

Biolayer Reference Reference Surface

R = 0.018/nm
Responsivity vs. Oxide Thickness
2.00 = 635 nm 1.50 Precent Modultion per nm 1.00 0.50 0.00 -0.50 -1.00 -1.50
ResponOx.qpc

-2.00 60 70 80 90 100 110 120 130 140 Oxide Thickness (nm)

2 nm

Antibody immobilization
8 Head Piezoelectric Printer
Spot Uniformity: +/- 2%

CCD Head Camera Photo from Printer

Rendered Interferometric Data Through PicoMapsTM Software

Spotted Disk Layout:

Spots: 100 radial 256 angular = 25,600 spots = 6,400 unit cells

70 pl drops 100 micron spots

Unit Cell Ag A Ag B

Disc Scan: 15,000 points per track @ 3.3 sec per point 8x averaging per track 1500 tracks @ 20 micron pitch Beam waist = 20 microns

Unit Cell

Data Analysis
Target Reference 1 mm

Post incubation

Pre incubation

Difference

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

Accuracy and Repeatability:

46pm pm 46 0.15

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

Probability Density

0.10

0.05

0.00 0.02

sdd.dat

0.04

0.06

0.08

0.10

0.12

0.14

0.16

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

(nm) Root Height Variance (pm)

BioCD Scaling Surface Sensitivity

2 = 0.2 pg/mm Sm = min As = hm w0
3 Integrated Waveguides

0.3 BioCD 0.1

Surface Plasmon Resonance

Sensit.qpc

0.03 30

100

300

1000

Height Root Variance (pm)

Assay Response Curve

1 QSSI-250 20 Hour Incubation 16% Biologically Active Change in Spot Mass 0.1

0.01 K = 35 ng/ml
D

0.001

Noise floor

Respon250.qpc

0.0001 0.1 1 10 100 Concentration (ng/ml)

Limit of Detection (LOD) scaling

100 Limit of Detection (LOD) [ng/ml]

10 2 ng/ml 1
k = 35 ng/ml
D

100 assys

200 pg/ml 10,000 assays

k = 10 ng/ml
D

0.1

act

= 17%

N = 0.1%

0.01

act

= 50%

N = 0.01%

0.001 100

Mx2

10

10

10

104

Number of Assays per Disc

Performance of Silicon BioCDs

(Jan. 25, 2007)

Property Limit of Detection

Scaling (expt. extrap.)

Selectivity Height Resolution (@ 20 )

Under assay conditions

Sensitivity (rand. noise)

At 1 mm2

Value 100 pg/ml single assay 10 ng/ml at 1000 assays 10 ng/ml in 7 mg/ml 20 pm 46 pm 0.2 fg/m 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 @ $100 per disc

Products & Manufacturing

BioCDs

Manufactured at Quadraspec, Inc. (West Lafayette, IN)

Manufactured at Medivative, Inc. (Indianapolis, IN)

Inspira SP250

Manufactured at Medivative, Inc. (Indianapolis, IN)

Inspira Lab Station

CHW Assay Dilution Study

Current Gold Standard
DiroChek Heartworm ELISA Serum Distribution
3500

Quadraspec
QCHW Heartworm Assay Serum Distribution

0.8
3000

0.7

0.6 A b so rb an ce at 630 n m

2500

Signal Value

0.5

2000

0.4

1500

0.3
1000

0.2
500

0.1

Negative Serum Samples

Positive Serum Samples

Negative Serum Samples

Positive Serum Samples

Detection at 1:35 Dilution

Detection at 1:270 Dilution

Molecular Interferometric Imaging (MI2)

Laser scanning vs. Full-field imaging

Laser CCD

Detector 635nm filter Objective 120nm SiO2 silicon wafer Objective 120 nm SiO2 silicon wafer White light

Spinner Laser scanning: Serial data acquisition Spatial resolution (20m) Works dry.

Translation/Rotation stage Full field imaging: Parallel data acquisition High spatial resolution. (0.5m) Works dry and wet

Microscope Image: 40x

3200 100 150 200 250 300 350 400 450 500 550 600 100 200 300 400 500 600 2500 2700 2800 3000 3100

2900

2600

QSSI-1861 120nm 40x 635 nm filter

Shearing In-Line Interferometry

Image 1

Image 2 Shift Wafer

Difference
Diff = 2

(I 2 I1 ) (I 2 + I1 )

0.02 400 0.015 500 0.01 0.005 0 700 -0.005 800 -0.01 -0.015 -0.02

600

900 300 400 500 600 700 800

QSSI-1861 120nm 40x

0.02 0.015 0.01 0.005 150 0 200 -0.005 250 -0.01 300 -0.015 -0.02

50

100

350

50

100

150

200

250

300

350

Scanning comparison
Full-Field
0.02 0.015 68 100 0.01 70 0.005 150 0 200 -0.005 250 -0.01 76 300 -0.015 78 50 100 150 200 250 300 350 -0.02 6680 6685 6690 6695 6700 6705 -0.03 -0.02 74 -0.01 72 0 0.01 0.02 66 50

Laser Scan
0.03

350

QSSI-903 120nm SiO2.

Mean height = 1.3 nm = 0.2 ML

QSSI-903

CV = 7%

QSSI-1370

Dome height = 0.7 nm

Ring height = 0.7 nm = 0.1 ML

QSSI-1709

Full-Field
Height Repeatibility (pm) 100

4x Objective 40x Objective

1 N

1/f Noise

20 pm/pixel
(0.5 m)
repeat.fullfield.qpc

10 10 100 Number of Averages

1000

Scaling Comparison: Laser Scanning:

hmin = 46 pm / pixel

S = 0.25 pg / mm

1 pixel = 20 m

Full-Field:
hmin = 60 pm / pixel
1 pixel = 0.5 m

S = 0.01 pg / mm

Scaling Comparison: Why so Good? Laser Scanning:

fsamp = 150 kHz

N = 16
BWFull R= = BWScan
Full N Full fsamp

Scan samp

10 7 1024 10 16
5

= 60

N Scan

Full-Field:
fsamp = 10 6 / 0.1sec = 10 MHz

N = 1024

BioCD

Thank You

Question 1

Question 2

Question 3

Question 4