U.S. patent application number 11/345566 was filed with the patent office on 2007-02-01 for differentially encoded biological analyzer planar array apparatus and methods.
Invention is credited to David D. Nolte, Leilei Peng, Fred E. Regnier, Manoj Varma, Ming Zhao.
Application Number | 20070023643 11/345566 |
Document ID | / |
Family ID | 37693293 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070023643 |
Kind Code |
A1 |
Nolte; David D. ; et
al. |
February 1, 2007 |
Differentially encoded biological analyzer planar array apparatus
and methods
Abstract
A method of probing a plurality of analyzer molecules
distributed about a detection platform is disclosed. The method
includes contacting a test sample to the plurality of analyzer
molecules, scanning the plurality of analyzer molecules at a rate
relating to a carrier frequency signal, and detecting the presence
or absence of a biological molecule based at least in part upon the
presence or absence of a signal substantially at a sideband of the
carrier frequency signal. A molecule detection platform including a
substrate and a plurality of targets positioned about the substrate
is also disclosed. Specific analyzer molecules adapted to bind a
specific analyte are immobilized about a first set of the targets.
Nonspecific analyzer molecules are immobilized about a second set
of the targets. The targets positioned about the substrate along at
least a segment of a scanning pathway alternate between at least
one of the first set and at least one of the second set. A method
including providing a substrate for supporting biological analyzer
molecules the substrate including at least one scanning pathway is
also disclosed. The scanning pathway includes a plurality of
scanning targets. Specific biological analyzer molecules adapted to
detect a specific target analyte are distributed about a first set
of the targets which alternate in groups of at least one with a
second set of the targets the second set of the targets not
including the specific biological analyzer molecules.
Inventors: |
Nolte; David D.; (Lafayette,
IN) ; Varma; Manoj; (West Lafayette, IN) ;
Regnier; Fred E.; (West Lafayette, IN) ; Peng;
Leilei; (West Lafayette, IN) ; Zhao; Ming;
(West Lafayette, IN) |
Correspondence
Address: |
BOSE MCKINNEY & EVANS LLP;JAMES COLES
135 N PENNSYLVANIA ST
SUITE 2700
INDIANAPOLIS
IN
46204
US
|
Family ID: |
37693293 |
Appl. No.: |
11/345566 |
Filed: |
February 1, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60648724 |
Feb 1, 2005 |
|
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|
60649070 |
Feb 1, 2005 |
|
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60755177 |
Dec 30, 2005 |
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Current U.S.
Class: |
250/288 |
Current CPC
Class: |
G01N 21/45 20130101;
G01N 21/553 20130101; G01N 21/645 20130101; G01N 33/54373
20130101 |
Class at
Publication: |
250/288 |
International
Class: |
H01J 49/00 20060101
H01J049/00 |
Goverment Interests
[0002] This invention was made with government support under grant
reference number NSF ECS-0200424 awarded by the National Science
Foundation. The Government has certain rights in the invention.
Claims
1. A method of probing a plurality of analyzer molecules
distributed about a detection platform comprising: contacting a
test sample to the plurality of analyzer molecules; scanning the
plurality of analyzer molecules at a rate relating to a carrier
frequency signal; and detecting the presence or absence of a
biological molecule based at least in part upon the presence or
absence of a signal substantially at a sideband of the carrier
frequency signal.
2. The method of claim 1 further comprising prescanning the
plurality of analyzer molecules before the contacting and improving
the detecting based upon a difference between the scanning and the
prescanning.
3. The method of claim 1 wherein the sideband is substantially free
from overlap with the carrier frequency signal.
4. The method of claim 1 wherein the detecting utilizes self
referencing phase quadrature interferometric detection.
5. The method of claim 1 wherein the detection platform is a
bio-CD.
6. The method of claim 1 further comprising suppressing the carrier
frequency signal.
7. The method of claim 1 wherein the detecting utilizes
interferometry and the scanning utilizes a laser beam.
8. The method of claim 1 further comprising detecting the presence
or absence of a second biological molecule based at least in part
upon the presence or absence of a second signal substantially at a
second sideband of the carrier frequency signal.
9. The method of claim 1 wherein the detecting includes detecting a
harmonic signal closest to zero frequency.
10. The method of claim 1 wherein the detecting includes detecting
a harmonic signal at a frequency greater than that of a harmonic
signal closest to zero frequency.
11. The method of claim 1 wherein the detecting includes detecting
a signal at or about a fundamental carrier frequency.
12. The method of claim 1 wherein the detecting utilizes
fluorescence detection.
13. A molecule detection platform comprising a substrate and a
plurality of targets positioned about the substrate wherein
specific analyzer molecules adapted to bind a specific analyte are
immobilized about a first set of the targets, and nonspecific
analyzer molecules are immobilized about a second set of the
targets, and the targets positioned about the substrate along at
least a first segment of a scanning pathway alternate between at
least one of the first set and at least one of the second set.
14. The platform of claim 13 wherein the targets positioned about
the substrate alternate along the first segment of the scanning
pathway between at least four of the first set and at least four of
the second set.
15. The platform of claim 13 wherein the platform is a micro
diffraction bio-CD, a phase contrast bio-CD, or an adaptive optics
bio-CD.
16. The platform of claim 13 wherein the nonspecific analyzer
molecules exhibit nonspecific background binding at least
substantially similar to the specific analyzer molecules.
17. The platform of claim 13 wherein the targets are
interferometric microstructures.
18. The platform of claim 13 wherein the targets positioned about
the substrate along at least a second segment of the scanning
pathway adjacent the first segment alternate between at least one
of the second set and at least one of the first set in the opposite
order as the alternation of the first segment.
19. The platform of claim 13 wherein the targets are substantially
contiguous along the segment of a scanning pathway.
20. A method comprising: providing a substrate for supporting
biological analyzer molecules the substrate including at least one
scanning pathway, the scanning pathway including a plurality of
scanning targets; and distributing specific biological analyzer
molecules adapted to detect a specific target analyte about a first
set of the targets which alternate in groups of at least one with a
second set of the targets, the second set of the targets not
including the specific biological analyzer molecules.
21. The method of claim 20 further comprising distributing
nonspecific analyzer molecules about the second set of the
targets.
22. The method of claim 20 wherein the first set of the targets
alternate in groups of at least four with the second set of the
targets.
23. The method of claim 20 further comprising: contacting a test
sample to the molecules; scanning the plurality of targets at a
rate; and detecting the presence or absence of a biological
molecule based at least in part upon the presence or absence of a
signal substantially about a frequency offset from a frequency
defined by the distribution of the targets and the scanning
rate.
24. The method of claim 23 wherein the detecting utilizes
fluorescence.
25. The method of claim 23 wherein the substrate is a surface of a
bio-CD, and the detecting utilizes phase quadrature interferometric
detection
Description
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Patent Application Serial No. 60/648,724, entitled "METHOD FOR
CONDUCTING CARRIER-WAVE SIDE-BAND OPTICAL ASSAYS FOR MOLECULAR
RECOGNITION," filed on Feb. 1, 2005 and the same is expressly
incorporated herein by reference.
TECHNICAL FIELD
[0003] The present invention generally relates to apparatus,
methods and systems for detecting the presence of one or more
target analytes or specific biological materials in a sample, and
more particularly to a laser compact disc system for detecting the
presence of biological materials and/or analyte molecules bound to
target receptors on a disc by sensing changes in the optical
characteristics of a probe beam reflected, transmitted, or
diffracted by the disc caused by the materials and/or analytes.
BACKGROUND
[0004] In many chemical, biological, medical, and diagnostic
applications, it is desirable to detect the presence of specific
molecular structures in a sample. Many molecular structures such as
cells, viruses, bacteria, toxins, peptides, DNA fragments,
pathogens, and antibodies are recognized by particular receptors.
Biochemical technologies including gene chips, immunological chips,
and DNA arrays for detecting gene expression patterns in cancer
cells, exploit the interaction between these molecular structures
and the receptors. [For examples see the descriptions in the
following articles: Sanders, G. H. W. and A. Manz, Chip-based
microsystems for genomic and proteomic analysis. Trends in Anal.
Chem., 2000, Vol. 19(6), p. 364-378. Wang, J., From DNA biosensors
to gene chips. Nucl. Acids Res., 2000, Vol. 28(16), p. 3011-3016;
Hagman, M., Doing immunology on a chip. Science, 2000, Vol. 290, p.
82-83; Marx, J., DNA Arrays reveal cancer in its many forms.
Science, 2000, Vol. 289, p. 1670-1672]. These technologies
generally employ a stationary chip prepared to include the desired
receptors (those which interact with the target analyte or
molecular structure under test). Since the receptor areas can be
quite small, chips may be produced which test for a plurality of
analytes. Ideally, many thousand binding receptors are provided to
provide a complete assay. When the receptors are exposed to a
biological sample, only a few may bind a specific protein or
pathogen. Ideally, these receptor sites are identified in as short
a time as possible.
[0005] One such technology for screening for a plurality of
molecular structures is the so-called immunological compact disk,
which simply includes an antibody microarray. [For examples see the
descriptions in the following articles: Ekins, R., F. Chu, and E.
Biggart, Development of microspot multi-analyte ratiometric
immunoassay using dual flourescent-labelled antibodies. Anal. Chim.
Acta, 1989, Vol. 227, p. 73-96; Ekins, R. and F. W. Chu,
Multianalyte microspot immunoassay--Microanalytical "compact Disk"
of the future. Clin. Chem., 1991, Vol. 37(11), p. 1955-1967; Ekins,
R., Ligand assays: from electrophoresis to miniaturized
microarrays. Clin. Chem., 1998, Vol. 44(9), p. 2015-2030].
Conventional fluorescence detection is employed to sense the
presence in the microarray of the molecular structures under test.
Other approaches to immunological assays employ traditional
Mach-Zender interferometers that include waveguides and grating
couplers. [For examples see the descriptions in the following
articles: Gao, H., et al., Immunosensing with photo-immobilized
immunoreagents on planar optical wave guides. Biosensors and
Bioelectronics, 1995, Vol. 10, p. 317-328; Maisenholder, B., et
al., A GaAs/AlGaAs-based refractometer platform for integrated
optical sensing applications. Sensors and Actuators B, 1997, Vol.
38-39, p. 324-329; Kunz, R. E., Miniature integrated optical
modules for chemical and biochemical sensing. Sensors and Actuators
B, 1997, Vol. 38-39, p. 13-28; Dubendorfer, J. and R. E. Kunz,
Reference pads for miniature integrated optical sensors. Sensors
and Actuators B, 1997 Vol. 38-39, p. 116-121; Brecht, A. and G.
Gauglitz, recent developments in optical transducers for chemical
or biochemical applications. Sensors and Actuators B, 1997, Vol.
38-39, p. 1-7]. Interferometric optical biosensors have the
intrinsic advantage of interferometric sensitivity, but are often
characterized by large surface areas per element, long interaction
lengths, or complicated resonance structures. They also can be
susceptible to phase drift from thermal and mechanical effects.
Current practice is to perform long time integrations (as in
fluorescence detection) to achieve a significant signal. However,
the long integration times place the measurement firmly in the
range of 1/f noise (frequency=1/.tau., where .tau. is the
measurement time). Likewise, SPR measurement approaches (for
example systems from Biacore) or resonant mirror approaches (for
example systems from SRU Biosystems) are angle resolved or
wavelength resolved, requiring detailed measurements that take long
integration times.
[0006] While the abovementioned techniques have proven useful for
producing and reading assay information within the chemical,
biological, medical and diagnostic application industries,
developing improved fabrication and reading techniques for planar
arrays with significant improvement in performance over existing
planar array technology is desirable.
SUMMARY
[0007] One embodiment according to the present invention includes a
method of probing a plurality of analyzer molecules distributed
about a detection platform. The method includes contacting a test
sample to the plurality of analyzer molecules, scanning the
plurality of analyzer molecules at a rate relating to a carrier
frequency signal, and detecting the presence or absence of a
biological molecule based at least in part upon the presence or
absence of a signal substantially at a sideband of the carrier
frequency signal.
[0008] Another embodiment according to the present invention
includes a molecule detection platform including a substrate and a
plurality of targets positioned about the substrate. Specific
analyzer molecules adapted to bind a specific analyte are
immobilized about a first set of the targets. Nonspecific analyzer
molecules are immobilized about a second set of the targets. The
targets positioned about the substrate along at least a segment of
a scanning pathway alternate between at least one of the first set
and at least one of the second set.
[0009] A further embodiment according to the present invention
includes a method including providing a substrate for supporting
biological analyzer molecules. The substrate includes at least one
scanning pathway. The scanning pathway including a plurality of
scanning targets. The method further includes distributing specific
biological analyzer molecules adapted to detect a specific target
analyte about a first set of the targets which alternate in groups
of at least one with a second set of the targets. The second set of
the targets does not include the specific biological analyzer
molecules.
[0010] Additional embodiments, aspects, and advantages of the
present invention will be apparent from the following
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows a graph of noise power density versus frequency
according to an embodiment of the present invention;
[0012] FIG. 2 shows a graph of power spectrum versus frequency
according to an embodiment of the present invention;
[0013] FIG. 3 shows a distribution of elements according to an
embodiment of the present invention;
[0014] FIG. 4 shows a distribution of elements according to an
embodiment of the present invention;
[0015] FIG. 5 shows scanning of an element according to an
embodiment of the present invention;
[0016] FIG. 6 shows a distribution of elements according to an
embodiment of the present invention;
[0017] FIG. 7 shows a distribution of elements according to an
embodiment of the present invention;
[0018] FIG. 8 shows a bio-CD according to an embodiment of the
present invention;
[0019] FIG. 9A shows a bio-CD according to an embodiment of the
present invention;
[0020] FIG. 9B shows a bio-CD according to an embodiment of the
present invention;
[0021] FIG. 10A shows a bio-CD according to an embodiment of the
present invention;
[0022] FIG. 10B shows a bio-CD according to an embodiment of the
present invention;
[0023] FIG. 11 shows a bio-CD according to an embodiment of the
present invention;
[0024] FIG. 12 shows scanning of elements according to an
embodiment of the present invention;
[0025] FIG. 13 shows a detection system according to an embodiment
of the present invention;
[0026] FIG. 14 shows a graph of time domain results of scanning a
differentially encoded MD-class calibration disk;
[0027] FIG. 15 shows a graph of frequency domain results of
scanning a differentially encoded MD-class calibration disk;
[0028] FIG. 16 shows a graph of frequency domain results of
scanning a differentially encoded MD-class disk;
[0029] FIG. 17 shows a graph of frequency domain results of
scanning a differentially encoded MD-class disk;
[0030] FIG. 18 shows a graph of frequency domain results of
scanning a differentially encoded MD-class disk;
[0031] FIG. 19 shows a graph of frequency domain results of
scanning a differentially encoded MD-class disk;
[0032] FIG. 20 shows a portion of an MD-class disk;
[0033] FIG. 21 shows a graph of time domain results of scanning the
disk of FIG. 20;
[0034] FIG. 22 shows a graph of frequency domain results of
scanning the disk of FIG. 20;
[0035] FIG. 23 shows a graph of time domain results of scanning a
PC-class disk;
[0036] FIG. 24 shows a portion of a PC-class disk;
[0037] FIG. 25 shows a magnified view of a portion of FIG. 24;
[0038] FIG. 26 shows Fourier domain results of scanning the disk of
FIG. 24;
[0039] FIG. 27 shows a demodulated image of the of the Fourier
domain results of FIG. 26;
[0040] FIG. 28 shows a graph of a comparison of prescan subtraction
without demodulation and prescan subtraction with demodulation.
DETAILED DESCRIPTION
[0041] For the purposes of promoting an understanding of the
principles of the invention, reference will now be made to the
embodiments illustrated in the drawings and specific language will
be used to describe the same. It will nevertheless be understood
that no limitation of the scope of the invention is thereby
intended, such alterations and further modifications in the
illustrated device, and such further applications of the principles
of the invention as illustrated therein being contemplated as would
normally occur to one skilled in the art to which the invention
relates.
[0042] With reference to FIG. 1 there is shown graph 1000 with
frequency increasing along its x axis as indicated by x axis arrow
1020 and noise power density increasing along its y axis as
indicated by its y axis arrow 1010. Frequency can be either
temporal frequency (Hz) or spatial frequency (1/cm). Graph 1000
illustrates noise power density versus frequency in the absence of
a carrier frequency. Curve 1030 illustrates the noise power density
of total noise as it varies with frequency. Curve 1040 illustrates
the noise power density of 1/f noise as it varies with frequency. A
bandwidth between frequencies 1060 and 1070 is indicated by arrows
BW. The total noise for this bandwidth is given by the area under
curve 1030 labeled 1080 which represents detected noise power for a
measurement taken at bandwidth BW. The frequency range where only
static is detectable is illustrated by arrows ST. The frequency
value of the 1/f noise knee is illustrated by line 1050 and
represents the frequency above which a signal may be detected over
noise.
[0043] With reference to FIG. 2 there is shown graph 2000 with
frequency increasing along its x axis as indicated by x axis arrow
2020 and power spectrum increasing along its y axis as indicated by
y axis arrow 2010. The power level of 1/f noise is illustrated by
curve 2030. A DC sideband signal 2040 having DC sideband center
frequency 2041, a carrier signal 2060 having carrier center
frequency 2061, and carrier sidebands 2050 and 2070 having carrier
sideband center frequencies 2051 and 2071, respectively, are also
shown.
[0044] Graph 2000 illustrates one example of frequency domain
detection of the molecular, cellular, or particulate content of a
liquid or air sample in which an analyte binds on or in a support
material to produce a periodic, quasi-periodic or harmonic
modulation of phase or amplitude of an electromagnetic wave that
probes the support material. The periodic or quasi-periodic
modulation can be in time or space, leading to a time-domain
carrier frequency or a space-domain carrier frequency, by relative
motion of the probe beam and support. The presence of the bound
analyte appears as a modulation sideband of the carrier frequency.
As shown in graph 2000, carrier sideband signals 2050 and 2070
indicate the presence of one or more target analytes bound to
analyzer molecules distributed about a support material which is
probed with an electromagnetic wave in a detection system. The
detection system preferably includes a photodetector, or another
detector responsive to electromagnetic waves, that outputs a
current as described below by Equation 1: i .function. ( t ) = 1 2
.times. ( 1 + cos .times. .times. .omega. c .times. t ) .times. ( 1
+ A .times. .times. cos .times. .times. .omega. m .times. t )
##EQU1## Equation 1 has a harmonic decomposition described by
Equation 2: i .function. ( t ) = .times. 1 2 + 1 2 .times. cos
.times. .times. .omega. c .times. t + A 2 .times. cos .times.
.times. .omega. m .times. t + A 4 .times. cos .function. ( .omega.
c + .omega. m ) .times. t + .times. A 4 .times. cos .function. (
.omega. c - .omega. m ) .times. t ##EQU2## Equation 2 describes a
DC sideband at .omega..sub.m, a carrier band at .omega..sub.c, and
two carrier sidebands at .omega..sub.c -.omega..sub.m and
.omega..sub.c+.omega..sub.m which correspond to DC sideband 2040, a
carrier 2060, and sidebands 2050 and 2070 as shown in graph 2000.
In Equations 1 and 2, t is time, i(t) is detector output current as
a function of time, .omega..sub.c is carrier angular frequency,
.omega..sub.m the modulation angular frequency, and A is the
envelope amplitude. In further embodiments detector output could be
a voltage, another electrical signal, an optical signal, or a
magnetic signal, for example, or some combination of these and/or
other outputs.
[0045] With reference to FIG. 3 there is shown a distribution of
elements 3000 including elements 3010 and 3020. Elements 3010 and
3020 are distributed about reading pathway 3004 which is defined on
a substrate. As shown by dashed lines 3030, 3040, 3050, 3060, and
3070, elements 3010 and 3020 are arranged in alternating groups of
four. As shown by ellipses 3006 and 3008 this pattern can continue
beyond the segment illustrated in FIG. 3 with the groups of four
elements alternating as described above. A unit cell includes a
group of four elements 3010 and a group of four elements 3020 as is
indicated by arrow UC between dashed lines 3030 and 3050. Scanning
footprint SF travels along reading pathway 3004 to scan the
distribution of elements 3000. Additional embodiments include
alternating groups of different numbers, for example, one, two,
three, five or more, and corresponding different sizes of unit
cells.
[0046] Elements 3010 include specific analyzer molecules which
selectively bind with a target analyte and elements 3020 include
nonspecific analyzer molecules which do not selectively bind with a
target analyte but may exhibit similar binding properties with
respect to other molecules. In a preferred embodiment according to
the present invention, elements 3010 include specific antibodies
immobilized about their surfaces, for example, as a monolayer,
fractional monolayer, partial monolayer, or near monolayer, and
elements 3020 include similarly immobilized nonspecific antibodies.
For example, if an assay is to be conducted to identify a
particular mouse protein the specific antibody could be goat
anti-mouse IgG (the antibody to the mouse protein produced by a
goat) and the nonspecific antibody could be goat anti-rat IgG (the
antibody to an analogous rat protein produced by a goat). The goat
anti-mouse IgG will selectively bind the mouse protein while the
goat anti-rat IgG will not bind with it or will have a
substantially lesser binding affinity, however, both IgGs exhibit
similar nonspecific background binding with molecules other than
the target analyte. In additional embodiments the non-specific
protein could be a non-IgG, for example, casein or bovine serum
albumin (BSA). These proteins could be used to test general
protein-protein background, and could be used to test for
systematics that are common to both groups of immobilized
molecules. In further embodiments the specific analyzer molecules
could be a cDNA that is complimentary to the target DNA, and the
non-specific group could be a statistically similar, but not
identical, cDNA. Additional embodiments cal include specific and
non-specific aptamers. A variety of other specific and nonspecific
antibody pairs may also be used, including those exhibiting varying
degrees of similarity in nonspecific background binding and those
not exhibiting similar nonspecific background binding. Furthermore,
combinations of specific and nonspecific analyzer molecules other
than antibodies may also be used. Additionally, nonspecific
analyzer molecules may be omitted entirely in which case elements
3020 would not include immobilized molecules. These alternative
exemplary embodiments and others can be used in connection with the
present embodiment and also in connection with the other
embodiments including those described elsewhere herein.
[0047] Distribution of elements 3000 is one example of differential
encoding or envelope modulation of bimolecular information.
According to a preferred embodiment of the present invention,
distribution of elements 3000 is on a bio-CD where elements 3010
and 3020 are interferometric microstructures formed on a surface of
the bio-CD, and reading pathway 3004 is one of a number of a
substantially concentric circular tracks. As described above,
elements 3010 on the track are active (carrying a specific
biological analyzer molecule) and elements 3020 are inactive
(carrying nonspecific molecules, no molecules, or inert molecules
that may be comparable in size with the analyzer molecule). In this
4 on/4 off format, the carrier frequency corresponds to the
positioning of each individual one of elements 3010 and 3020, and
the detection frequency corresponds to the repeat period of the
unit cell UC which is every eight elements. Thus, the detection
frequency is equal to one-eighth of the carrier frequency. At disk
rotation speeds of 6000 rpm (100 Hz) and 1024 elements per track,
the carrier frequency is approximately 100 kHz and the detection
frequency is approximately 12.5 kHz. A wide variety of other
bimolecular platforms, scanning rates, and element distributions
including, for example, those described herein, are contemplated
and can result in a variety of other carrier frequencies and
detection frequencies.
[0048] According to a preferred embodiment of the present
invention, an optical detection system including two phase-locked
loops in series, with the front end referenced to the carrier
frequency, and the back end referenced to the unit cell can be used
to scan a bio-CD having distribution of elements 3000 with a laser.
Differential encoding of distribution of elements 3000, for example
as described above and elsewhere herein, can preferably reduce
susceptibility to laser intensity drift or disk wobble by
subtracting out these and other system drifts and biases, and can
preferably directly subtract non-specific background binding, for
example if the off region is printed with nonspecific antibody. One
example of a detection system according to a preferred embodiment
of the present invention can be found in U.S. Pat. No. 6,685,885
which is hereby incorporated by reference. This detection system
could also be any other detection system responsive to
electromagnetic waves including for example those described
elsewhere herein.
[0049] According to a preferred embodiment of the present invention
the detection system can utilize phase quadrature interferometric
techniques. Examples of phase quadrature interferometric techniques
include the micro-diffraction quadrature class ("MD-class") and
adaptive optic quadrature class ("AO-class") as described in U.S.
application Ser. No. 10/726,772 filed on Dec. 3, 2003 entitled
"Adaptive Interferometric Multi-Analyte High-Speed Biosensor"
(published on Aug. 26, 2004 as U.S. Pub. No. 2004/0166593), the
contents of which are incorporated herein by reference. Other
examples of phase quadrature interferometric techniques include the
phase-contrast quadrature class ("PC-class") as described in U.S.
Provisional Patent Application No. 60/649,070, filed Feb. 1, 2005,
entitled "Phase-Contrast Quadrature For Spinning Disk
Interferometry And Immunological Assay", U.S. Provisional Patent
Application No. 60/755,177, filed Dec. 30, 2005, entitled
"Phase-Contrast BioCD: High-Speed Immunoassays at Sub-Picogram
Detection Levels", and U.S. Application Serial No. ______ being
filed the same day as the present application that claims priority
to these two provisional applications and entitled "Method And
Apparatus For Phase Contrast Quadrature Interferometric Detection
Of An Immunoassay." The disclosure of the utility application being
filed on the same day as the present application is incorporated
herein by reference. Additionally, further embodiments of the
present invention include detection systems adapted to utilize
surface plasmon resonance or SPR, fluorescence, resonance and other
techniques in which high frequency modulation in time or space
originates from analyte bound to a solid support with a spatial
frequency that is scanned to produce a sideband indicating the
presence of the analyte. Still other preferred embodiments of the
present invention include detection platforms for use in these and
other detection systems which include distributions of targets
including analyzer molecules which produce sideband signals that
depend upon modulation indicative of the presence of an
analyte.
[0050] With reference to FIG. 4 there is shown a biosensor platform
4000 including a substrate 4030 having an upper surface 4010 and
lower surface 4020. Interferometric elements 4040, 4050, 4060, and
4070 are formed on the upper surface 4010 of substrate 4030.
Platform 4000 may also include additional interferometric elements
in addition to those shown in the portion of platform 4000
illustrated in FIG. 4. A laser beam 4002 having wavelength .lamda.
scans the interferometric elements 4040, 4050, 4060, and 4070 in
the direction indicated by arrow DM. Elements 4040 and 4050 include
specific analyzer molecules immobilized about their scanned
surfaces and elements 4060 and 4070 include nonspecific analyzer
molecules immobilized about their scanned surfaces. These specific
and nonspecific analyzer molecules can be, for example, the same or
similar to those described above in connection with FIG. 3 and
elsewhere herein. This configuration of specific and nonspecific
analyzer molecules of biosensor platform 4000 is another example of
differential encoding according to a preferred embodiment of the
present invention. In one preferred embodiment of the present
invention platform 4000 is a micro-diffraction bio-CD and elements
4040, 4050, 4060, and 4070 are radial spokes distributed about the
surface of the bio-CD. Platform 4000 can also be any of various
other biosensor platforms including, for example, those described
herein.
[0051] Biosensor platform 4000 is one example of carrier
suppression according to a preferred embodiment of the present
invention. Elements 4060 and 4040 have a height illustrated by
arrows HA and elements 4050 and 4070 have a height illustrated by
arrows HB. Height HA is about .lamda./8 and height HB is about
3.lamda./8. Successive scanning of elements alternating between
height HA and HB flips the phase quadratures detected for
successive elements. This results in a modulation at about twice
the amplitude as compared to a platform having interferometric
elements with substantially uniform element heights. The carrier is
suppressed by an approximately .pi. phase difference between phase
quadrature signals detected for successive elements. Carrier
suppression may be useful in a variety of circumstances. In one
example, where carrier side bands are weak relative to the carrier,
carrier noise can impact detection. In another example where
carrier sidebands overlap with the carrier, carrier noise can also
impact detection. Carrier wave suppression can preferably increase
the ratio of signal to noise. Complete carrier suppression or
double sideband detection may be used to improve the signal to
noise ratio of detection in these and other situations. Partial
carrier suppression may also improve the signal to noise ratio of
detection in these and other situations. Carrier wave suppression
can also be accomplished in other manners, for example, fabrication
of disk structures and reflectivities relative to beam width,
through use of a clipper circuit that clips the high signal
detected from a land of a detection platform, or through use of a
filter, for example a band stop filter.
[0052] With reference to FIG. 5 there is shown an example of a
scanning 5000 during which footprint 5020 passes over element 5010.
Areas 5021 are the areas of the scanning footprint not over element
5010 and area 5011 is the area in which scanning footprint 5020
overlaps element 5010. According to a preferred embodiment element
5010 is a gold microdiffraction element placed on a partially
reflecting substrate. This embodiment allows carrier suppression by
the total power reflected from the element being equal to the total
power reflected under the condition of quadrature which removes the
large modulation caused by the approximately 50% amplitude
modulation of a micro diffraction bio-CD. This effect can be
illustrated through the following equations. The total electrical
(far) field is given by Equation 3: E T = E 0 A .function. [ r L
.times. A L + r r .times. A r .times. e i .times. .times. .PHI. ]
##EQU3## The total reflected intensity is given by Equation 4: I T
= E 0 2 A .function. [ R L .times. A L 2 + R r .times. A r 2 + 2
.times. r L .times. r r .times. A L .times. A r .times. cos .times.
.times. .PHI. ] ##EQU4## Under the condition of Land: .PHI.=0,
A.sub.L=A and A.sub.r=0. Thus, intensity reflected by land is given
by Equation 5: I.sub.L=I.sub.0R.sub.L Under the condition of
Quadrature: .PHI.=.pi./2. Thus, the reflected intensity under a
condition of quadrature is given by Equation 6: I Q = E 0 2 A
.function. [ R L .times. A L 2 + R r .times. A r 2 ] = I 0
.function. [ R L .times. a L 2 + R r .times. a r 2 ] ##EQU5## where
a.sub.i is the area fraction, and a.sub.L+a.sub.r=1. Conditions of
balanced operation are given by Equations 7 and 8: I.sub.Q=.sub.L
R.sub.L.alpha..sub.L.sup.2+R.sub.r.alpha..sub.r.sup.2=R.sub.L The
solution of which are given in Equations 9 and 10: 1 - a L 1 + a L
= R L R r ##EQU6## a L = 1 - R L R r 1 + R L R r ##EQU6.2## For
Equations 3-10, I.sub.r is the total reflected intensity, I.sub.L
is the intensity reflected by land, I.sub.O is the incident
reflected intensity, I.sub.Q is the reflected intensity under a
condition of quadrature, E.sub.o is the reflected field, A is the
total area, A.sub.L is area 5021, A.sub.r is area 5011, a.sub.L is
A.sub.L divided by the area of the beam footprint, a.sub.R is
A.sub.L divided by the area of the element 5010 intersecting
element 5020, R.sub.L is |r.sub.L|.sup.2, R.sub.r is
|r.sub.r|.sup.2 and .PHI. is the phase difference between reflected
components of the laser. Thus, if the partially reflective
substrate is silicon, for example, which has R.sub.L=32% and
R.sub.r=98%, then a.sub.L=51% and a.sub.r=49%.
[0053] With reference to FIG. 6 there is shown a biosensor platform
6000 including substrate 6030 having an upper surface 6010 and a
lower surface 6020. Upper surface 6010 includes analyzer molecules
6040, 6050, 6060, 6070, 6080 and 6090 immobilized about surface
6010. Analyzer molecules 6040, 6060, and 6080 are specific analyzer
molecules for selectively binding a particular analyte and analyzer
molecules 6050, 6070 and 6090 are nonspecific analyzer molecules.
The specific and nonspecific analyzer molecules can be, for
example, the same or similar to those described elsewhere herein.
FIG. 6 shows one example of an alternating pattern of specific and
nonspecific analyzer molecules. Laser beam 6002 scans the analyzer
molecules in the direction indicated by the arrow DM which is
preferably accomplished by rotating the platform 6000 but could
also be accomplished by other movement of platform 6000 or by
movement of beam 6002. According to a preferred embodiment of the
present invention platform 6000 is a phase contrast bio-CD or an
adaptive optical bio-CD and analyzer molecules 6040, 6050, 6060,
6070, 6080 and 6090 are radial spokes or other patterns of analyzer
molecules, however, platform 6000 could also be another kind of
bio-CD or other platform including, for example, those described
elsewhere herein.
[0054] During scanning of platform 6000 by laser beam 6002 signal
phase modulation depends only upon the binding differences between
the specific and nonspecific analyzer molecules. For example,
nonspecific binding that is common to both the types of analyzer
molecules is not imparted onto the signal beam or has minimal
impact on the signal beam. The detected signal is therefore
independent of nonspecific binding. In this embodiment there is no
signal detected at or about the carrier frequency and only the
modulation caused by binding of the specific analyte and the
specific analyzer molecule is detected. This is one example of
differential encoding including carrier wave suppression and double
sideband detection.
[0055] With reference to FIG. 7 there is shown a biological
analyzer platform 7000 including substrate 7030 including upper
surface 7010 and lower surface 7020. Interferometric elements 7070
are distributed about upper surface 7010 and are spaced apart by
gaps 7060. Interferometric elements 7070 include specific
biological analyzer molecules 7040 and nonspecific biological
analyzer molecules 7050 immobilized about their surfaces which can
be the same or similar to those described elsewhere herein. Groups
of the interferometric elements and analyzer molecules 7090 and
7091 are also shown. Groups 7090 and 7091 have patterns of specific
and nonspecific analyzer molecules that are at spatial frequencies
with a .pi. phase difference, that is, the positions of specific
and nonspecific analyzer molecules are flipped between groups 7090
and 7091. Platform 7000 is preferably an adaptive optical bio-CD,
however, platform 6000 could also be any other type of biosensor
platform or another type of bio-CD including, for example, those
described elsewhere herein.
[0056] During scanning of platform 7000 by a laser beam the phase
of the carrier is periodically flipped by .pi. for successive
groups 7090 and 7091. The effect of the phase flipping of the
carrier is that the carrier is suppressed in the power spectrum and
the modulation due to binding of a specific analyzer molecule to
the specific antibodies is detectable at carrier sidebands. This is
one example of differential encoding including carrier wave
suppression and double sideband detection.
[0057] According to a preferred embodiment modulated signals are
detected within a detection bandwidth .DELTA.f.sub.d. Narrow
bandwidths reject more noise, but the detection bandwidth should
preferably not be smaller than the signal bandwidth, otherwise a
part of the signal is rejected with the noise. The signal bandwidth
is determined by the relationship described by Equation 11:
.DELTA..omega..sub.8.DELTA..tau.=1 where
.DELTA..omega..sub.s=2.pi..DELTA.f.sub.s, .DELTA.f.sub.s is the
signal bandwidth, and .DELTA..tau. is the duration of either a
contiguous part of the signal, or the duration of the signal
detection measurement. In preferred embodiments utilizing bio-CDs,
the carrier frequency, f.sub.carrier, is set by the rotation
frequency of the bio-CD, f.sub.disk, and by the number of spokes,
targets, or interferometric elements, N, around a specified
circumference as described by Equation 12: f.sub.carrierNf.sub.disk
The signal bandwidth .DELTA.f.sub.s is described by Equation 13:
.DELTA. .times. .times. f s = f disk 2 .times. .pi. ##EQU7## The
relative signal bandwidth .DELTA.f.sub.rel is described by Equation
14: .DELTA. .times. .times. f rel = .DELTA. .times. .times. f f
carrier ##EQU8## For a single continuous track around a
circumference, the relative bandwidth .DELTA.f.sub.rel is described
by Equation 14: .DELTA. .times. .times. f rel = 1 2 .times.
.pi..DELTA..tau. .times. .times. f carrier = f disk 2 .times. .pi.
.times. .times. Nf disk = 1 2 .times. .pi. .times. .times. N
##EQU9## If a circumference is divided into S equal arcs of M
spokes, the relative bandwidth increases by a factor of S as
described by Equation 15: .DELTA. .times. .times. f rel S = N M
.times. .DELTA. .times. .times. f rel = S .times. .times. .DELTA.
.times. .times. f rel ##EQU10## Thus, for example, if N=1024, and
S=16, the relative bandwidth is 0.25%. If f.sub.disk=100 Hz, then
f.sub.s=100 kHz, .DELTA.f.sub.s=16 Hz and .DELTA.f.sub.s rel=256
Hz. These relations suggest that S up to 128 segments or more is
clearly a possible scenario for homogeneous bandwidths for which
.DELTA.f.sub.s=2 kHz and .DELTA.f.sub.s rel=2%.
[0058] The foregoing example describes the case of homogeneous
signal bandwidth. Signal bandwidths in practice are generally
larger than the homogeneous bandwidths. These arise, for example,
from frequency instability, which in the bio-CDs is from
inhomogeneities in the fabricated or printed spokes. If the
placement of the spokes is only accurate to 10 microns, then the
bandwidth of the repetitive spoke pattern is approximately 4 kHz
with a relative bandwidth of 4%. This inhomogeneous signal
bandwidth sets the correct detection bandwidth for the bio-CDs. The
number of segments can be increased to increase the homogeneous
bandwidth until it is equal to the inhomogeneous bandwidth to the
relationships described by Equations 16 and 17:
.DELTA.f.sup.S=.DELTA.f.sub.in hom BW= {square root over
(2)}f.sub.in hom For detection bandwidth BW, this sets the maximum
segment number according to Equation 18: S = 2 .times. .pi. .times.
.times. N .function. ( BW f carrier ) ##EQU11## which for BW=3 kHz
and f.sub.carrier=100 kHz for N=1024, this sets the maximum
S=136.
[0059] The ability to support segments suggests a disk array layout
that segments the printed antibodies into wells. For N wells on a
disk or S segments, the size of a well and its radial thickness are
given by Equations 19 and 20: a = r .times. .times. d .times.
.times. .theta. .times. .times. dr = r .times. .times. 2 .times.
.pi. S .times. dr = A / N ##EQU12## dr = AS 2 .times. .pi. .times.
.times. rN = ( R 2 2 - R 1 2 ) .times. S 2 .times. .times. rN
##EQU12.2## where a is the area of a well, r is radius, dr is
radial thickness of a well, .theta. is angular position, d.theta.
is well arclength, A is the area of the annular region between
radii R.sub.2 and R.sub.1, N is number of wells, S is the number of
segments, R.sub.1 is the inner radius, and R.sub.2 is the outer
radius.
[0060] With reference to FIG. 8 there is shown a bio-CD 8000
according to one embodiment of the present invention. Bio-CD 8000
is a 100 mm diameter disk or silicon wafer, however, any other
dimension disk, wafer chip or other substrate or platform could
also be used. Bio-CD 8000 includes sectors 8001, 8002, 8003, 8004,
8005, 8006, 8007, 8008, 8009, 8010, 8011, 8012, 8013, 8014, 8015,
and 8016. Bio-CD 8000 further includes substantially concentric
tracks of wells 8021, 8022, 8023, 8024, 8025, 8026, 8027, and 8028.
Bio-CD 8000 has S=16 sectors, N=128 then T=8 (tracks) and the inner
track radius and radial thicknesses are given in Table I:
TABLE-US-00001 Inner Track Radius Radial Thickness dr Track Number
(millimeters) (millimeters) 8028 20 6.56 8027 26.56 4.94 8026 31.50
3.60 8025 35.10 3.39 8024 38.50 3.13 8023 41.63 2.93 8022 44.56
2.76 8021 47.33 2.62
Bio-CD 8000 is one example of an equal area well layout according
to the present invention. Other layouts are also contemplated, for
example, a 512 well layout with S=16, T=32, and any other
combination of sectors and tracks. According to a preferred
embodiment layouts are used which bring the aspect ratio of
arclength and radial thickness closer to unity which simplifies
fabrication. Fabrication of this and other embodiments of the
present invention can include particular features for various
classes of bio-CDs. For example, a micro-diffraction bio-CD can
have radial spokes fabricated from gold across the entire disk, and
wells defined by hydrophobic dams. A pin plotter or ink-jet printer
modified from biochip array printers can be used to deposit an
equal amount of analyzer molecules into each well. Different
antibodies can be deposited which then self-immobilize on thiolated
gold. In another example gel printing can be used. In another
example, for adaptive optical bio-CDs and phase constant bio-CDs,
spokes can be printed as inert protein, dams can be put into place
and antibody deposited into the wells by pin array plotters or
protein spotters.
[0061] With reference to FIGS. 9A, 9B, 10A, 10B and 11 there are
shown bio-CDs 9000A, 9000B, 10000A, 10000B, and 11000 according to
embodiments of the present invention where the wells are of equal
area. In these embodiments, dr is held constant among the tracks,
and ds=rd.theta. is also held constant. This leads to a varying
d.theta. across the disk. In the preferred embodiment where well
areas remain are equal, the radial width of each well is constant
which simplifies design of the protein plotter, and optimal use of
real-estate is made. This embodiment requires a carrier spoke
number C to vary with radius, also causing the carrier frequency to
vary with radius (for constant angular velocity). The relation of
the spoke number is given by Equation 21: C = 2 .times. .pi.
.times. .times. r .LAMBDA. ##EQU13## where .LAMBDA. is the spatial
period, usually .LAMBDA.=2w, where w is the beam waist. For a beam
waist of 20 microns and .LAMBDA.=40 microns, this gives the number
of spokes as a function of radius C=3000 at r=20 mm and C=8000 at
r=50 mm. The carrier frequencies are 300 kHz and 800 kHz,
respectively.
[0062] For N wells, the area of each well is given by Equation 22:
.alpha.=rd.theta.dr=A/N The aspect ratio a.sub.r is set by the
Equation 23: rd.theta.=.alpha..sub.rdr The radial widths and
angular widths are given by Equation 24: dr = A a r .times. N
.times. .times. and ##EQU14## d .times. .times. .theta. = 1 r
.times. a r .times. A N ##EQU14.2## FIG. 9A shows a 96 well disk
with an aspect ratio of 1 and dr=7.5 mm, a=61 mm.sup.2, T=4,
S.sub.i=15, and S.sub.o=33. FIG. 9B shown a 96 well disk with an
aspect ratio of 4 and dr=4.3 mm, a=64 mm.sup.2, T=7, S.sub.i=8, and
S.sub.o=19. The well in FIGS. 9A and 9B areas are approximately 0.6
cm.sup.2. FIG. 10A shows a 512 well disk with an aspect ratio of 4,
dr=1.76 mm, a=12.7 mm.sup.2, T=17, S.sub.i=17, and S.sub.o=42, FIG.
10B shows a 1000 well disk with an aspect ratio of 4, dr=1.25 mm,
a=6.4 mm.sup.2, T=24, S.sub.i=24, and S.sub.o=59. FIG. 11 shows an
8000 well disk with an aspect ratio of 4, dr=0.45 mm, a=0.82
mm.sup.2, T=66, S.sub.i=69, and S.sub.o=172. A variety of other
disks with equal area wells and unequal well areas are also
contemplated. In general, larger aspect ratios have narrower
detection bandwidth, but more tracks with smaller track
pitches.
[0063] With reference to FIG. 12 there are shown examples of
scanning targets 12000. Targets 12000 are a periodically
alternating pattern of targets including specific antibodies 12010
and targets including nonspecific antibodies or not including
antibodies 12020. Specific and nonspecific antibodies are being
immobilized about a substrate, for example, as described herein.
After exposure to a sample including a specific target analyte,
targets 12010 have the analyte bound to their analyzer molecules
while targets 12020 exhibit little or no binding of the specific
analyte. The period of the alternating pattern is shown by arrows
LL, and the spatial frequency of the pattern is inversely
proportional to its period as shown by Equation 25: v spatial = 1
.LAMBDA. ##EQU15## where .LAMBDA. is the spatial periodicity and
.nu..sub.spatial is the spatial frequency.
[0064] During scanning targets 12000 are illuminated by a scanning
footprint such as a laser spot. The scanning footprint could be,
for example, focused laser spot vv which has a width w.sub.o less
than spatial periodicity .LAMBDA. (preferably
w.sub.o<<.LAMBDA.) and moves relative to the targets 12000
with a velocity in the direction indicated by arrow v. Under these
scanning conditions the spatial frequency .nu..sub.spatial is
converted into temporal frequency on the transmitted or reflected
beam as described by Equation 26: f=Vv where f is the carrier
frequency of phase or amplitude modulation.
[0065] The scanning footprint could also be, for example, broad
area laser spot z which has a width w.sub.o greater than spatial
periodicity .LAMBDA. (preferably w.sub.o>>.LAMBDA.) and can
be stationary or can move relative to the targets 12000 with a
velocity V in the direction indicated by arrow v. When laser spot z
is stationary and broadly illuminates the spatial frequency, then
the spatial frequency leads to diffraction at specific angles as
described by Equation 27: .theta. = sin - 1 .function. ( .lamda.
.LAMBDA. ) ##EQU16## where .lamda. is the illumination wavelength,
and .LAMBDA. is the spatial period. When laser spot z moves over to
targets 12000, or targets 12000 move with velocity V, then the
diffracted orders acquire a phase modulation that is
time-periodic.
[0066] The foregoing examples illustrate how spatial frequencies on
a scanning platform, for example a chip or disk, can be converted
into temporal frequencies in a laser scanning system, and how the
two types of frequencies can be combined when a laser probes more
than one target on the platform.
[0067] With reference to FIG. 13 there is shown detection system
13000 which includes detector 13010 and detector 13020. Detectors
13010 and 13020 could be any detectors for detecting
electromagnetic waves, for example optical detectors. System 13000
further includes probe beam 13030 which can be a focused probe beam
or a broad area probe beam. Probe beam 13030 scans targets 13040
which move relative to beam 13030 with a relative velocity in the
direction indicated by arrow RV. The scanning targets 13040 by beam
13030 results in a transmitted or reflected mode 13012 and a
diffracted mode 13022. Mode 13012 is directed to detector 13010 and
mode 13022 is directed to detector 13020. Reference beam 13023 is
directed to detector 13010 and reference beam 13023 is directed to
detector 13020. Reference beam 13023 is preferably maintained in a
condition of phase quadrature relative to the transmitted mode
13012. Reference beam 13033 is preferably maintained in a condition
of phase quadrature relative to diffracted mode 13022. System 13000
also includes beam splitters 13011 and 13021 which could also be
adaptive optical beam combiners. Having a reference wave that is in
phase quadrature with detected signal allows a small shift in the
phase modulation of the signal to linearly proportional change in
detected intensity allowing signal modulation per bound analyte
molecule to be maximized. Reference beams 13033 and 13023 can be
added before photodetectors or can be combined adaptively with
signals. Reference beams 13033 and 13023 can arise from a
diffracted spatial mode, for example, in the case of wavefront
splitting, from free space, or from partial reflections, for
example, in the case of amplitude splitting. It is also
contemplated that detection system 13000 could include only one or
the other of detectors 13010 and 13020 and their related beams and
modes.
[0068] Experimental demonstrations of several exemplary embodiments
including carrier side band detection according to the present
invention will now be described in connection with FIGS. 14-28.
With reference to FIG. 14, there is shown graph 14000 with time
increasing along its x axis as indicated by x axis arrow 1420 and
signal intensity (voltage) increasing along its y axis as indicated
by y axis arrow 14010. Graph 14000 further shows signal 14030 which
is a voltage signal that varies with time. Signal 14030 results
from the scanning of an MD-class calibration disk which was
fabricated with 1024 gold spokes deposited radially on a dielectric
substrate. The average (mean) spoke height was 80 nm. Of the 1024
spokes, 512 spokes were below the average height, 512 spokes were
above the average height, and the spokes alternated between those
above the average height and those below the average height.
[0069] Scanning the MD-class calibration disk produced signal 14030
which includes a series of alternating local minima 14031 and 14032
corresponding to and indicating the two spoke heights. The signal
intensity difference between the alternating local minima 14031 and
14032 is illustrated by arrow 14040 and corresponds to a height
difference of about 30 nm between alternating spokes. This height
difference is representative of the height difference cause by
certain target analytes to analyzer molecules. The signal level
corresponding to the average spoke height of about 80 nm is
indicated by dashed line 14050. The MD-class calibration disk thus
provides a simulation of a differential encoding scheme whereby
every other alternating spoke includes analyzer molecules that bind
a target analyte and can be compared to a reference spoke. The fast
relative comparison between the two types of spokes allows for
significant noise reduction.
[0070] With reference to FIG. 15 there is shown graph 15000 with
frequency increasing along its x axis as shown by x axis arrow
15020 and power increasing logarithmically along its y axis as
shown by y axis arrow 15010. Graph 15000 shows the frequency domain
results of the scanning of the MD-class calibration disk described
above in connection with FIG. 14. Graph 15000 shows carrier signal
15030 at 200 kHz, sideband signal 15031 at 100 kHz, and sideband
signal 15032 at 300 kHz. Thus the sideband signals are present at
half carrier frequency increments. A strong 1/f noise peak 15040 is
present at zero frequency, and a significantly suppressed noise
floor is present at the frequencies of carrier and sideband signals
15030, 15031 and 15032. The noise suppression by operating at this
scanning rate is over 60 dB or 3 orders of magnitude better signal
to noise ratio when compared to a static measurement at DC (zero
frequency). This is a fundamental advantage to high speed
repetitive sampling according to certain embodiments of the present
invention.
[0071] With reference to FIG. 16 there is shown graph 16000 with
frequency increasing along its x axis as shown by x axis arrow
16020 and power increasing along its y axis as shown by y axis
arrow 16010. Graph 16000 shows an example of protein side-band
detection for an MD-class disk having proteins (in this case
antibody IgG molecules) immobilized on a 1024-spoke disk with 64
segments composed of 8 elements with protein and 8 elements
without. This created a disk with an alternating pattern of 8 gold
spokes carrying protein followed by 8 bare gold spokes. This
pattern repeated for a total of 64 segments each with a total of 16
elements divided into 8 with protein and 8 without. The proteins
were patterned using a polydimethylsiloxane (PDMS) stencil on the
disk. A control track which did not include printed protein was
also included on the disk. The results of scanning the control
track are indicated by dotted line 16060 and the results of
scanning a track including the patterned protein are indicated by
line 16050.
[0072] Graph 16000 shows 16030 the 1/f noise at DC and two DC
sideband signals 16031 and 16032. A carrier frequency signal (not
shown) is present at about 100 kHz. The presence of protein is
detected as a 1/64 harmonic of the carrier frequency at about 1.6
kHz as shown by signal 16032 and also by signal 16031 at about -1.6
kHz. A second harmonic signal 16034 and 16033 is also present at
1/32 the carrier frequency and is caused by slight asymmetry in the
deposition of the proteins. A comparison of protein track signal
16050 and signal 16060 of a control track containing no protein
illustrates the strong effect of the protein in producing sideband
signals with a 20:1 signal to noise ratio as indicated by arrow
16040.
[0073] With reference to FIG. 17 there is shown graph 17000 with
frequency increasing along its x axis as shown by x axis arrow
17020 and power spectrum increasing logarithmically along its y
axis as shown by y axis arrow 17010. Graph 17000 presents average
values for scanning of six tracks of the MD-class disk which is
described above in connection with FIG. 16. Graph 17000 shows a
comparison of 1/64 harmonic signal 17040 at about 1.6 kHz, which is
generated by and indicates the presence of protein, and carrier
signal 17030. As illustrated by arrow 17050, the protein modulation
is about 4.6% of the carrier wave, which is consistent with a
monolayer of immobilized protein.
[0074] With reference to FIG. 18 there is shown graph 18000 with
frequency increasing along its x axis as shown by x axis arrow
18020 and power spectrum increasing logarithmically along its y
axis as shown by y axis arrow 18010. While the side bands off of DC
yielded the best signal-to-noise ratio for scanning the MD-class
disk described above in connection with FIG. 16, every carrier
harmonic includes two side-bands. Thus, as shown in graph 18000
fundamental carrier harmonic 18030 which is at about 80 kHz
includes sidebands 18031 and 18032. Sidebands 18031 and 18032 are
small peaks above and below the harmonic carrier frequency 18030
which indicate the presence of the protein. Every other carrier
harmonic also has two associated sidebands.
[0075] With reference to FIG. 19 there is shown graph 19000 with
frequency increasing along its x axis as shown by x axis arrow
19020 and power spectrum increasing logarithmically along its y
axis as shown by y axis arrow 19010. Graph 19000 shows carrier
frequency harmonics 19030A (which is the first carrier harmonic
18030 at about 80 kHz described above in connection with FIG. 18),
19030B, 19030C, 19030D, 19030E, 19030F, and 19030G. Each carrier
harmonic includes protein sidebands, though the wide frequency
range of the graph 1900 makes it difficult to see the protein
sidebands for all the harmonics. Graph 19000 also demonstrates the
noise-floor roll-off for high frequencies associated to the transit
time t=w.sub.0/v of a point on the disk across the width of the
focused laser spot w.sub.o. Line 19050 shows the approximate
midpoint of the noise floor roll off.
[0076] With reference to FIG. 20 there is shown a fluorescence
microscope image of portion of an MD-class disk 20000 according to
one embodiment of the present invention. Disk 20000 is a
half-harmonic differentially encoded MD-class disk which was
created using photolithography to immobilize protein on every
alternating spoke. During this process half the spokes were covered
by photo-patterned photoresist while the other half were exposed to
protein. The photoresist was then removed to uncover bare gold
spokes. This results in a disk where protein is immobilized on
every alternating spoke as shown by lines 20010 (indicating
deposition of specific antibody) and 20020 (indicating no
deposition of antibody, or deposition of a non-specific antibody).
The width of each protein deposit is about 20 microns as indicated
by arrows SW. This half-harmonic differential encoding in which
every alternating spoke carries protein results in the highest
signal-to-noise ratio being attained. This provides for the
highest-frequency differencing measurements, and also boosts the
total protein signal when the zero-frequency upper sideband and the
carrier frequency lower side-band merge into a single sideband half
way between DC and the fundamental carrier frequency.
[0077] With reference to FIG. 21 there is shown graph 21000 with
time increasing along its x axis as shown by x axis arrow 21020 and
voltage increasing along its y axis as shown by y axis arrow 21010.
When a 512 differential encoded disk is rotated and scanned, the
protein modulates the gold spokes with a frequency at half the
fundamental carrier frequency. Graph 21000 shows the detected time
trace 21030 from a 512 differential encoded disk. Trace 21030 shows
an alternating pattern between the bare and protein-carrying spokes
as indicated by the minimum points trace 21030 which alternate in
amplitude at the rate of a half harmonic signal 21040.
[0078] With reference to FIG. 22 there is shown graph 22000 with
frequency increasing along its x axis as shown by x axis arrow
22020 and power spectrum increasing logarithmically along its y
axis as shown by y axis arrow 22010. Graph 22000 shows the
frequency domain side band effect of the disk described above in
connection with FIG. 21. The half-frequency harmonic protein signal
22040 is strong and occurs near the frequency of lowest noise
between DC signal 22050 and the first carrier signal 22030. As
shown in graph 22000 the DC sideband and first carrier sidebands
have merged at the half-frequency harmonic protein signal 22040.
Furthermore, the protein signal 22050 itself has sidebands 22041
and 22042 caused by slight asymmetries in the protein printing. The
signal-to-noise ratio is greatest in this situation where the noise
floor is lowest. Thus, detection of protein at signal 22040
represents the optimal performance condition for carrier sideband
detection on the MD-class disk described above.
[0079] With reference to FIG. 23 there is shown graph 23000 with
frequency increasing along its x axis as shown by x axis arrow
23020 and power spectrum increasing logarithmically along its y
axis as shown by y axis arrow 23010. Graph 23000 shows the power
spectrum for an embodiment of a PC-class disk with a periodic
pattern of protein on a dielectric disk with no other disk
structure. Graph 23000 shows DC signal 23040 and protein signal
23030 which is caused by and indicates the presence of protein. For
this PC-class embodiment, the carrier frequency is attributable
entirely to the protein, without any contribution from
microstructures or other physical structures on the disk. The
detection of periodic patterns of immobilized protein on a flat
surface is one example of carrier-wave suppression that was
discussed above. Additional embodiments including, for example,
suppressing the carrier of the gold spokes on MD-class disks are
also discussed above. Analyzer molecule patterns on PC-class disks
offer a embodiment of side-band detection and manipulation that
significantly improves the sensitivity of the bio-CD because the
periodic protein patterns can themselves be modulated to form
larger spatial patterns.
[0080] With reference to FIG. 24 there is shown a portion of a
patterned protein PC-class disk 24000 according to one embodiment
of the present invention. The radial direction is in the vertical
direction the angular direction around the disk is in the
horizontal direction. As shown in FIG. 11, the portion of disk
24000 is in a checkerboard pattern. Substantially rectangular areas
of periodic stripes of protein 24010 are alternated with
substantially rectangular areas of bare disk 24020. Each
substantially rectangular area has a radial distance of
approximately 0.5 mm indicated by arrow RD and an angular distance
of approximately 45 degrees indicated by arrow AD. The height of
the printed protein stripes is approximately 5 nm. The signal
resulting from scanning the PC-class disk is differential, showing
only the steps up and down from the protein stripes.
[0081] With reference to FIG. 25 there is shown a magnified portion
25000 of the PC-class disk 24000 shown in FIG. 24 individual
protein bands 24011 of protein regions 24010 are visible in
magnified portion 25000. The rectangular spatial patterns of areas
24010 and 4020 of disk 24000 create sidebands on the protein peak
in the power spectrum. The long-range spatial patterns can be
detected using a sideband demodulation process conceptually similar
to the demodulation of FM radio. The long-range protein patterns
constitute an envelope that modulates the carrier wave. By
demodulation, the envelope is extracted. Because it is more slowly
varying, envelope demodulation makes it possible to perform more
accurate prescan subtraction.
[0082] An exemplary procedure for sideband detection will now be
described with reference to FIGS. 26, 27 and 28. FIG. 26 shows an
isolated protein peak 26030 in the power spectrum. The horizontal
axis 26010 is temporal frequency and the vertical axis 26020 is
spatial frequency along the radius of the disk. The sub-peaks 26031
and 26032 represent the long-range envelope pattern. To demodulate
the signal and extract the protein envelope, this protein peak is
shifted back to DC and then Fourier-transformed back into the space
domain. The resulting demodulated image is shown in FIG. 27. Only
the long-range checkerboard pattern 27000 corresponding to areas
24010 and 24020 is visible, with the periodicity of the individual
protein bands 24011 removed. After demodulation, subtracting a
prescan becomes much more accurate.
[0083] FIG. 28 shows graph 28000 with distribution probability on
the vertical axis and height in nm on the horizontal axis. Graph
28000 shows the results of subtracting a prescan from a postscan
before demodulation as distribution 28010 and performing the same
subtraction after demodulation as distribution 28020. The error in
height reduces from 75 pm for distribution 28010 to 20 pm for
distribution 28020 with demodulation. This increased accuracy
improves surface mass sensitivity by over a factor of 3 in this
example.
[0084] While the examples illustrated and described above in
connection with FIGS. 14-28 have made reference to particular
embodiments, for example, MD-class disks with protein attached
using photolithographic techniques and PC-class disks with printed
protein, these specific embodiments are merely exemplary and it is
contemplated that differential encoding and sideband detection
described above could be employed with a variety of other
embodiments according to the present invention including those
described elsewhere herein.
[0085] Various embodiments according to the present invention can
include a variety of biosensor platforms including those described
above. For example, these platforms include bio-CDs such as
micro-diffraction bio-CDs, adaptive-optical bio-CDs, phase-contrast
bio-CDs, and others. Details relating to these various classes of
bio-CDs can be found, for example, in the aforementioned patents
and patent applications. These platforms further include bio-chips,
immunological chips, gene chips, DNA arrays, platforms used in
connection with fluorescence assays and other platforms and
substrates supporting planar arrays including analyzer molecules
including, for example, those described herein.
[0086] Various embodiments according to the present invention can
include a variety of analyzer molecules useful in detecting the
presence or absence of a variety of target analytes in a solution
to be tested. For example, these analyzer molecules can include
antibodies or immunoglobulins, antigens, DNA fragments, cDNA
fragments, aptameres, peptides, proteins, and other molecules.
Various embodiments according to the present invention can include
combinations of one or more of the foregoing and other types of
analyzer molecules known to a person of ordinary skill in the art
arranged, for example, in a planar array.
[0087] Various embodiments according to the present invention can
be used in connection with a variety of scanning and detection
techniques. For example, such techniques include interferometry,
including surface normal interferometry techniques, and preferably
phase quadrature interferometry techniques where one detected
optical mode differs in phase from another by about .pi./2 plus or
minus about twenty percent or an odd integer multiple thereof,
and/or self referencing interferometry techniques where a reference
wave is generated locally with respect to a signal wave so that the
reference and signal waves experience common aberrations and path
length changes and thus maintain a constant relative phase without
the need for active stabilization of different light paths,
florescence techniques and platforms, resonance techniques and
platforms, and other techniques and platforms.
[0088] As used herein terms relating to properties such as
geometries, shapes, sizes, physical configurations, speeds, rates,
frequencies, periods, amplitudes, include properties that are
substantially or about the same or equal to the properties
described unless explicitly indicated to the contrary.
[0089] While the invention has been illustrated and described in
detail in the drawings and foregoing description, the same is to be
considered as illustrative and not restrictive in character, it
being understood that only preferred embodiments have been shown
and described and that all changes and modifications that come
within the spirit of the invention are desired to be protected.
* * * * *