48 Optics & Photonics News October 20041047-6938/04/10/0048/6-$0015.00 © Optical Society of America

The BioCDSpinning-Disk Interferometry

David D. Nolte and Fred E. Regnier

High speed

interferometry

on a spinning

disk opens new

opportunities

for sensitive

material

metrology.

One promising

application is

fast molecular

screening in

diagnostic

medicine.

October 2004 Optics & Photonics News 49

wave-front-splitting null interferometer,which can be understood by consideringFig. 1. By carefully selecting the focusinglens, a Gaussian laser beam can be madeto straddle the pit (ridge) with half theintensity falling on the ridge and half onthe land. If the height of the ridge is �/4,then the phase of the field reflected fromthe ridge is � out of phase relative to thefield reflected from the land. In the farfield, along the optic axis, the two fieldsinterfere destructively, producing a nullcondition at the detector.

As the laser scans a track of pits, theintensity alternates between full inten-sity reflected from the land, and zerointensity reflected while straddling aridge. These are the two states thatenable digital recording. From Fig. 1 it is clear why fingerprints have so littleeffect on digital CDs: small changes inpit depth in the land or null states pro-duce only a small quadratic change in therecorded intensity.

The challenge of the spinning disk isto overcome a hostile mechanical envi-ronment: vibrations and wobble make itdifficult to extract in reflection any signal

with a stable average phase, which wouldseem to preclude the possibility of per-forming interferometry. Overcoming this challenge is part of the genius ofCompaan’s original idea for the CD. Byilluminating the pit and land by means ofa single spatial optical mode, the wave-front-splitting interferometer is self-ref-erencing: the signal and reference wavestravel identical paths through the opticalsystem all the way to the detector. Thisexplains why you can jog and still listento your Discman while it performs a mil-lion null interferometric measurementsper second.

Spinning-disk interferometry (SDI)The condition of quadrature is an impor-tant alternative that allows the digital CDto be converted into an analog CD that ismaximally sensitive to small phase differ-ences. In Fig. 1, when the ridge height isequal to �/8, the ridge and land signalshave a relative phase of �/2. In this casethe intensity response curve has a maxi-mum slope, which produces the largestintensity modulation per change in the

Figure 1. Wave-front-splitting interferometry on a gold microstructured ridge. The far-field intensity along the optic axis follows a sinusoidal interferometric response as afunction of height h. Destructive interference occurs for a ridge height of �/4 (used indigital CDs), while maximum linear sensitivity to an added material thickness �h isachieved for the quadrature conditions at h = �/8 and 3�/8.

h

w0

w

BioCD Substrate

GaussianBeam

δhland

"pit"

0.0

0.2

0.4

0.6

0.8

1.0

Ridge Height (h)

λ/8 λ/4 3λ/8 λ/20

Land

DigitalCD

Quad. 1 Quad. 2

T he BioCD is a biological compactdisc. It is printed with proteinsinstead of digital “ones” and

“zeros.” It reads molecular recognitioninstead of music. The operation of theBioCD is derived from that of a conven-tional CD, except for the fact that itoperates as an analog sensor rather thanas a digital recording medium. Whiledigital CDs are relatively immune to thepresence on their surface of fingerprints,the analog BioCD is exquisitely sensitiveto submonolayers of immobilized pro-tein. The BioCD is one example of thebroader concept of spinning-disk inter-ferometry (SDI), in which intrinsicallystatic measurements (such as molecularrecognition) are converted into high-frequency optical modulation.1 As abiosensor, the BioCD may one day bedeveloped into a fast, cheap and reliablemultianalyte assay for clinical medicineand environmental testing.

Compact disc technologyThe digital compact disc was invented in1969 by Klaas Compaan, a Dutch physi-cist working for Philips Corporation. In

1970, he and Pete Kramer developedthe first glass disc prototype

and began using a laser toachieve diffraction-limited

spot sizes for the read-out.2 The goal was simple: to replace the

musical LP—with itsgrooves and mechanical

(analog) scratches—with adisc that could be recorded dig-

itally with ones and zeros. Thesimplest format is a disk that reflects

either high intensities or low intensitiesto represent the two states. The lowreflectance state is produced by scatteringlight off a small pit. A CD is simply a col-lection of tracks of pits recessed from thesurface (called the land) that are createdby embossing a plastic substrate with aglass master. The pits are interrogatedby means of a laser focused from theopposite (unembossed) side of the poly-carbonate disk, making the indentationsappear as ridges, but the “pit” terminol-ogy is still often used.

There are approximately three billionpits, on 22 thousand tracks, on a digitaldata CD. Each pit acts as an individual

Inte

rfer

omet

ric In

tens

ity

optical phase of the ridge. In the quadra-ture condition, the spinning disk takesstatic structure (surface roughness,index variations, etc.) and turns it into high-frequency phase modulation thatcan be detected with narrow bandwidthfar from 1/f noise. We can apply substan-tial optical knowledge to the problemsince detection of high-frequency phase modulation is common in interferometry.3

The spinning disk is a generalizedoptical modulator that operates in a waysimilar to a mechanical chopper, exceptfor the fact that it produces small-signalamplitude and/or phase modulationinstead of large amplitude modulation,as would be the case, for example, with a fixed-blade chopper wheel. Takentogether, small phase modulation andoptical phase quadrature make it is possi-ble to operate the device in the linearsmall-signal limit. This makes it a sensi-tive analog measurement device of broadusefulness.

The SDI concept makes possibleapplications that seek to measureextremely small variations in the extinc-tion coefficient and refractive index of amaterial. The technique can be used tomeasure the thickness or refractive indexvariations of the spinning disk itself. Or itcan be used to measure the thickness andrefractive index of a material deposited in

a spatially periodic pattern on the disk. Inthe case of an unpatterned disk, the mod-ulation caused by heterogeneity of thedisk causes dynamic light scattering.4

When the disk is used as a substrate thatsupports a periodic material structure,dynamic light scattering defines the noisefloor for the measurement.

Potential applications of spinning-diskinterferometry span the fields of materialscience (material metrology), microbiol-ogy (viruses and bacteria), clinicalmedicine (immunoassays), homelandsecurity (biological and chemical warfaredetection), environmental science (waterand air monitoring) and food science(contamination), among others. The central concept of SDI is so basic—conversion of an intrinsically static

measurement into a high-frequency opti-cal modulation—that many more appli-cations are likely to emerge. Our currentprinciple interest is the BioCD.

The BioCDThe BioCD is a spinning-disk, self-refer-encing interferometer that has a layer ofantibodies deposited on top of its ridges.Antibodies are recognition moleculesthat bind strongly to one specific protein(called their antigen) but do not bind toany other molecules. Different antibodiesbind to different antigens, and biotech-nology can be used to produce antibodiescapable of recognizing a broad range ofpossible analytes. For this reason, clinicalassays (such as the PSA test used to detectthe prostate-specific antigen) are oftenbased on antibody specificity and arecalled immunoassays.5

The goal of the BioCD is to take thehighly static problem of molecular recog-nition (slow molecule-molecule bind-ing)—ordinarily carried out by detectingfluorescent changes incurred at a fixedspot—and converting the problem to oneof high-frequency phase modulation.

When an antibody film of height �h isattached to the ridge in Fig. 1, it modifiesthe far-field diffraction. The far-fielddiffraction for the three conditions ofland, quadrature and null of Fig. 1 aresimulated in Fig. 2(a); the change in

50 Optics & Photonics News October 2004

SPINNING-DISK INTERFEROMETRY

Figure 2. (a) Calculated far-field diffraction for three ridge heights h = 0 (land), �/8 (quadrature) and �/4 (null). (b) Relativechange in diffraction caused by an extra optical phase �� = 4�(n-1)�h/� for ridge relative to the land. A strong linear responseoccurs in quadrature.

The spinning-disk interferometry concept

makes possible applications that seek to measure extremelysmall variations in theextinction coefficient and refractive index

of a material.

�I

I(�)

� (radians)� (radians)

-0.08 -0.06 -0.04 -0.02 0 0.02 0.04 0.06 0.08-0.06 -0.04 -0.02 0 0.02 0.04 0.06

0.010

0.005

0.000

-0.005

-0.010

-0.015

-0.020

-0.025

1.00

0.75

0.50

0.25

0.00

(a)

quadrature

quadrature

Ridge Height0�/8�/4

0�/8�/4

Ridge Height

(b)

October 2004 Optics & Photonics News 51

diffraction is shown in Fig. 2(b) when therelative phase between the ridge and theland is changed by �� = 4�(n-1)�h/�where �h = 8 nm and n = 1.3, appropriatefor a monolayer of antibodies. Thequadrature condition produces a 4.7 per-cent change in the diffracted signal.6 Atquadrature, the change is linear in phase,but in the land and null conditions it isquadratic in phase. This makes it possiblefor small submonolayers to be detected inquadrature, but not in the null interfer-ometer configuration.

BioCD quadrature classesThere is more than one way to performself-referencing interferometry: all thatis required is a reference and a signalwave that can be phase locked. Figure 3shows two types of self-referencing spin-ning-disk interferometry defined bytheir quadrature mechanisms: micro-diffraction quadrature (using ridges as discussed above) and adaptive opticalquadrature (using two-wave mixing in the far field between the signal and a reference beam inside a photo-refractive film).

The trade-off between the two classesof BioCD is the trade-off between thenear- and the far-field. The micro-diffraction approach requires detailedmicrostructuring of the disk and carefulmechanical tracking by the objective lens,but detection in the far field case is sim-ply by means of an apertured photode-tector. The adaptive optical approach hassimple disk fabrication and low require-ments on head tracking, but requires asophisticated adaptive optical mixer inthe far field. The adaptive approach tendsto be more robust, while the micro-diffraction approach tends to have highersensitivity. The technical details of thetwo quadrature classes are discussed next.

Microdiffraction quadratureThe diffraction of the probe beam fromthe microstructured ridge plays the roleof a wave-front-splitting interferometersimilar to Young’s double slit experiment.Just as in the case of any two-wave inter-ferometer, the diffracted intensity in thefar field along the optic axis has a simplesinusoidal dependence on the ridgeheight. In this particular case, however,the higher-angle diffraction plays the role

of the conjugate port, in which the inter-ferometric response has opposite sign.For this reason the far-field diffractionpattern must be apertured to pass thecentral diffraction peak, while higherangles are blocked.

To facilitate selective molecule immo-bilization chemistry on the disk, wedeposit a pattern of radial spokes made of gold ridges on a high-reflectance sub-strate (either a silicon wafer or a lasermirror). In this case, tracks are defined bythe selection of the radial distance to thelaser spot and require no active tracking.As the disk spins, the laser repetitivelysamples the land and the quadrature condition, as shown in Fig. 4 for an

immunoassay carried out againstimmunoglobulin.5 At quadrature, thereflected intensity is approximately 50 percent. When a single antibody layeris immobilized on the gold spokes, thephase of the ridge changes and the inten-sity at quadrature drops. When the trackis exposed to a non-target protein (in thiscase, a rabbit protein), no binding occurs,and the quadrature signal remains mostlyunaltered, indicating a negative test to anon-specific antigen. However, when thetrack is subsequently exposed to the spe-cific protein (antigen) that is bound bythe antibody, the signal at quadraturedrops further, indicating a positive testto specific antigen.7

SPINNING-DISK INTERFEROMETRY

Figure 3. The two BioCD self-referencing quadrature classes trade-off complexitybetween the near and far fields. In microdiffraction quadrature, the disk is microstruc-tured to produce a far-field diffraction pattern that is apertured to retain the zero orderonly. In adaptive optical quadrature, the disk is simply printed with protein in a spokepattern, and the signal and reference phases are locked in the far field by two-wavemixing in a photorefractive quantum well device.

BioCD Quadrature Classes

MicrodiffractionQuadrature

Far-Field

Near-Field

Protein on MicrostructuredGold Spokes

Aperture

Printed Protein

Adaptive OpticalQuadrature

PRQW

More accurate measurement can bemade with the BioCD by changing fromtime-domain experiments to frequency-domain experiments with much nar-rower detection bandwidths. This changealso allows us to acquire dose responsecurves for the BioCD. The results are acombination of sensitivity, selectivity and dynamic range, which are indicatedin Fig. 5.

The figure shows two dose responsecurves as raw voltage signal vs. appliedanalyte concentration from a real-timeapplication of the analyte to the spinningdisk.9 The sample is centrifuged off thedisk within approximately 10 seconds,a time period which represents the incu-bation time for each assay. The signal forspecific antigen has an onset at 10 ng/ml(sensitivity) and saturates at 1 mg/mlwith a dynamic range of 50:1. Theresponse curve for a non-specific anti-gen has no discernable onset up to 1 mg/ml, a value which sets a lowerbound for the selectivity of theimmunoassay at levels greater than10,000. This number can be roughlytranslated into the number of equivalent-concentration analytes simultaneouslypresent in a sample that can be analyzed

without cross-talk. Alternatively, thisnumber can be translated into the con-centration range that can be assayedbetween two analytes. In this case, a non-specific analyte can be 10,000 times moreconcentrated than the specific analyteand still not cause false positive readings.

Adaptive optical quadratureLocking the relative phase between thereference and signal waves in the far fieldis an attractive option because it facili-tates disk fabrication and tracking. Thebeams can, for instance, be mixed by useof adaptive interferometry.10-13 The mix-

ing is accomplished inside a photorefrac-tive quantum well (PRQW) device thatperforms as a sensitive dynamic holo-graphic film.14 The phase is locked by themutual diffraction off the dynamicallyupdating holographic grating; quadra-ture is selected by the choice of operatingwavelength for the laser source.13 For the adaptive BioCD, protein is printeddirectly onto glass substrates in a spokepattern with a spoke width larger thanthe focal waist of the laser beam. As the disk spins, the printed protein (andthe protein that binds to it) impart a repetitious phase modulation on the signal beam.

Slow phase modulation from mechan-ical vibration and disk wobble is com-pensated by the dynamic holograms,while the high-frequency phase modula-tion of the spinning proteins passesthrough to the detector as intensitymodulation.

The protein-printed BioCD disk isincubated (while the disk is not spinning)with different samples around concentrictracks. In our first multi-analyte assay, weprinted spokes of protein A and back-filled the surface with protein B. Thisproduced an alternating spoke pattern,

52 Optics & Photonics News October 2004

SPINNING-DISK INTERFEROMETRY

Figure 4. Time trace of a BioCD signal. High reflectance is offthe land, while low reflectance is the quadrature condition whenthe laser spot straddles a ridge (red trace). When a layer of anti-bodies is added selectively to the ridge, the signal drops (blacktrace). Exposing the antibodies to a non-specific protein inducesno significant change (dashed green trace), but when the anti-bodies are exposed to and bind a specific antigen, the signaldrops further (blue trace).8

Figure 5. Dose response curve for a microdiffraction quadratureBioCD. The onset of selective binding to a specific immunoglobu-lin (IgG) protein is at 10 ng/ml and saturates above 1 mg/ml. Thenon-specific binding has no onset below 1 mg/ml which translateinto a chemical specificity of greater than 10,000.

Nor

mal

ized

Sig

nal

Bind

ing

Sign

al (

�V)

Time (A.U.)

Protein Concentration (g/ml)

Land

Quadrature

-200 -150 -100 -50 0 50 100 150 200

10-10 10 -8 10 -6 10 -4 10 -2 100

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3

200

150

100

50

0

-50

More accurate measurement can bemade with the BioCD

by changing from time-domain experiments

to frequency-domainexperiments with much narrower

detection bandwidths.

Specific ProteinOther Protein

DynamicRange(50:1)

Sensitivity(10 ng/ml)

Selectivity

(>04)

October 2004 Optics & Photonics News 53

on the same disk, of the two types of pro-teins. The spokes were exposed to specificantibody A in tracks 2 and 3 and mea-sured, and then exposed to antibody B intracks 3 and 4 and measured. The finalresults are displayed in Fig. 6 as two-dimensional (2D) frequency-radius plots.

The data show a strong signal at thecarrier frequency of the spokes for theradii incubated with a single specific anti-body. Track 1 shows a strong response toantibody A and track 3 shows a strongresponse to antibody B. Track 2, exposedto both antibodies, shows no signalbecause both antibody A and antibody B bind to the spokes and cancel out thephase-modulated signal.

These experiments demonstrate theusefulness of adaptive optics applied toan immunoassay, but considerable workis necessary to demonstrate the same levels of sensitivity and selectivity thathave already been demonstrated by the microdiffraction BioCDs. Micro-diffraction is simpler, and much closer in concept to conventional CDs.However, the stability of the adaptiveapproach lends itself more to fundamen-tal quantitative research.

Future prospectsIt is now understood that one’s state ofhealth depends on complex protein net-works in which concentrations of scoresor even hundreds of proteins are inter-connected and dependent on each other,often with nonlinear kinetics varyingperson to person.

For instance, the blood is a kaleido-scopic soup of molecules: hormones,proteins, DNA, sugars, fats, antibodies—over a thousand different types ofmolecules coming from all parts of thebody. Every cell, every tissue, every organof the body contributes its own charac-teristic mix of molecules to the blood.Each mixture is like a signature or a fingerprint. To gain a more completeunderstanding of where the state ofhealth of an individual is today andwhere it will be next year, or how it willrespond to a drug, requires an analyticalassay like the BioCD that can test forhundreds or thousands of analytes.

The challenge for such a global assaytechnology is to measure hundreds or

thousands of trace concentrations withhigh specificity and large dynamicrange—which we have already begun todemonstrate. For point-of-care testing itis also necessary for the technology tobe fast, cheap and reliable. If the BioCDcan satisfy these requirements, it couldreplace the current paradigm of clinicaltesting in outside labs and bring testingto the point of care in doctor’s offices andin the home, to become a powerful toolfor diagnostic and preventive medicine.

AcknowledgmentThis work was supported by the NationalScience Foundation grant ECS-0200424.

David D. Nolte (nolte@physics.purdue.edu) andFred E. Regnier are with Purdue University. The

author has had a long-standing interest inadaptive interferometry and is a Fellow ofOSA and APS.

References

1. M. Varma et al., “High-Speed Label-Free Multi-AnalyteDetection through Micro-interferometry,” in

Microarrays and Combinatorial Technologies forBiomedical Applications: Design, Fabrication, andAnalysis, 4966, D. V. Nicolau and R. Raghavachari,eds.(SPIE, 2003), 58-64.

2. K. C. Pohlmann, The Compact Disc Handbook, 2nd ed., Madison, Wisconson (A-R Editions Inc., 1992).

3. C. B. Scruby and L. E. Drain, Laser Ultrasonics:Techniques and Applications, (Adam Hilger, 1990).

4. B. J. Berne and R. Pecora, Dynamic Light Scattering:With Applications to Chemistry, Biology, and Physics(Dover, 2000).

5. D. Wild, “The Immunoassay Handbook,” GrovesDictionary, 2001.

6. M. Varma et al., “Multi-Analyte Array Micro-DiffractionInterferometry,” in Microarrays: Design, Fabricationand Reading, 4626, B. J. Bornhop et al., eds., (SPIE,2002) 69-77.

7. M. M. Varma et al., Biosens. Bioelectron. 19, 1371-6(2004).

8. M. M. Varma et al., Opt. Lett. 29, 950-2 (2004).

9. M. Varma et al., “Spinning-Disk Interferometry forDetection of Antigen-Antibody Recognition: TheBioCD,” submitted to Anal. Chem. 2004.

10. R. K. Ing and J.-P. Monchalin, Appl. Phys. Lett. 59,3233-5, 1991.

11. P. Delaye et al., J. Opt. Soc. Am. B 14, 1723-34, 1997.

12. A. Blouin and J.-P. Monchalin, Appl. Phys. Lett. 65,932-4, 1994.

13. D. D. Nolte et al., J. Opt. Soc. Am. B 18,195-205,2001.

14. D. D. Nolte, J. Appl. Phys., 85, 6259, 1999.

SPINNING-DISK INTERFEROMETRY

Figure 6. Adaptive BioCD 2-analyte binding experiment. All tracks were dual printedwith mouse antigen and rabbit antigen. Tracks 2 and 3 were incubated with anti-rabbitIgG, and Tracks 3 and 4 were incubated with anti-mouse IgG. Selective binding isobserved in Tracks 2 and 4, while in Track 3 the two signals cancel.

Member

Track Radius

Frequency (kHz)

Track 5

Track 4

Track 3

Track 2

Track 1

Control

Anti Mouse

Anti Mouse +Anti Rabbit

Anti Rabbit

Control

30 40 50 60 70