U.S. patent application number 14/452316 was filed with the patent office on 2015-03-05 for polarization based interferometric detector.
The applicant listed for this patent is Bioptix Diagnostics, Inc.. Invention is credited to John Hall, Oyvind Nilsen, Viatcheslav Petropavlovskikh.
Application Number | 20150062593 14/452316 |
Document ID | / |
Family ID | 38157121 |
Filed Date | 2015-03-05 |
United States Patent
Application |
20150062593 |
Kind Code |
A1 |
Hall; John ; et al. |
March 5, 2015 |
POLARIZATION BASED INTERFEROMETRIC DETECTOR
Abstract
A sensor and method for determining the optical properties of a
sample material is disclosed. The sensor comprises a light source
that generates a linearly polarized light beam having a
predetermined polarization orientation with respect to the plane of
incidence. The linearly polarized light beam is reflected off the
sample and is split into second and third light beams where the
second and third light beam consist of the combined projections of
mutually orthogonal components of the first light beam. A signal
processor measures the intensity difference between the second and
third light beams to calculate the phase difference induced by the
sample material.
Inventors: |
Hall; John; (Broomfield,
CO) ; Petropavlovskikh; Viatcheslav; (Louisville,
CO) ; Nilsen; Oyvind; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bioptix Diagnostics, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
38157121 |
Appl. No.: |
14/452316 |
Filed: |
August 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13929731 |
Jun 27, 2013 |
8830481 |
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14452316 |
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11735900 |
Apr 16, 2007 |
8488120 |
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13929731 |
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11379026 |
Apr 17, 2006 |
7233396 |
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11735900 |
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Current U.S.
Class: |
356/491 |
Current CPC
Class: |
G01N 21/45 20130101;
G01N 2021/212 20130101; G01N 21/552 20130101; G01N 21/41 20130101;
G01N 21/553 20130101; G01N 21/253 20130101; G01N 21/21
20130101 |
Class at
Publication: |
356/491 |
International
Class: |
G01N 21/45 20060101
G01N021/45; G01N 21/552 20060101 G01N021/552; G01N 21/21 20060101
G01N021/21 |
Claims
1-73. (canceled)
74. A device for detecting a sample material, comprising: a. an
optical element that comprises or is adjacent to an interface
having a surface configured to come in contact with said sample
material; b. a light source that generates a first tight beam for
application to said optical element and towards said interface,
wherein said first light beam comprises a first polarization
component and a second polarization component, and wherein upon
application of said first light beam to said optical element, ent,
said first light beam is reflected away from said interface with a
phase shift in said first tight beam having a sensitivity to
surface refractive index change of at least about 5.times.10.sup.-8
Refractive Index Units (RIU) to permit detection of at least about
50 femtograms of said sample; c. at least one optical retarder that
provides substantially circular polarization in said first tight
beam; d. a beam splitter downstream of said interface that splits
said first tight beam into a second light beam and a third light
beam after said first light beam is reflected away from said
interface; e. a detector module downstream of said polarizing beam
splitter, wherein said detector module accepts and generates
signals from said second tight beam and said third light beam; and
f. a processor programmed to calculate a phase shift between said
first and second polarization components based on said signals
generated from said second and third light beams, to permit the
detection of at least about 50 femtograms of said sample.
75. The device of claim 74, wherein said first polarization
component and second polarization component are substantially in
phase relative to each other.
76. The device of claim 74, wherein said first polarization
component and said second polarization component are substantially
orthogonal to each other.
77. The device of claim 74, wherein said optical element comprises
an optically dense material.
78. The device of claim 74, wherein said phase shift is a variable
phase shift between said first polarization component and said
second polarization component.
79. The device of claim 74, wherein said light source comprises a
linearly polarized coherent light.
80. The device of claim 79, wherein said linearly polarized
coherent light has a wavelength in the range of about 500
nanometers to 700 nanometers.
81. The device of claim 79, wherein said linearly polarized
coherent light is oriented at a predetermined angle with respect to
the plane of incidence of said interface.
82. The device of claim 81, wherein said predetermined angle is
based on the maximum phase shift between said first and second
polarization components due to a change adjacent to said
surface.
83. The device of claim 74, wherein said surface comprises a
material that has a higher index of refraction than said
sample.
84. The device of claim 74, wherein said surface comprises a
material including glass, plastic, silicon or ceramic.
85. The device of claim 74, wherein said detector module comprises
(i) a first detector that measures an intensity of said second
light beam and (ii) a second detector that measures an intensity of
said third light beam.
86. A method for detecting a sample material, comprising: a
directing a first light beam comprising a first polarization
component and a second polarization component towards an optical
element that comprises or is adjacent to an interface having a
surface configured to come in contact with said sample material; b.
reflecting said first light beam away from said interface with a
phase shift in the first light beam having a sensitivity to surface
refractive index change of at least about 5.times.10.sup.8
Refractive Index Units (RIU) to permit detection of at least about
50 femtograms of said sample, which first light beam has
substantially circular polarization; c. splitting said first light
beam into a second light beam and third light beam after said first
light beam is reflected away from said interface; d. generating
signals from said second and third light beams; and e. calculating
a phase shift between said first and second polarization components
based on said signals generated from said second and third light
beams, thereby detecting at least about 50 femtograms of said
sample.
87. The method of claim 86, wherein said sample material comprises
nucleic acid, viral particles, bacteria or organic molecules.
88. The method of claim 86, wherein signals from said second and
third light beams are generated separately.
89. The method of claim 86, wherein said first polarization
component and said second polarization component are substantially
in phase and orthogonal relative to each other.
90. The method of claim 86, wherein said phase shift is a variable
phase shift between said first polarization component and said
second polarization component.
91. The method of claim 86, wherein said first light beam is a
linearly polarized coherent light beam that is rotated at a
predetermined angle with respect to a plane of incidence of said
interface.
92. The method of claim 91, wherein said linearly polarized
coherent light beam has a wavelength in the range of about 500
nanometers-700 nanometers.
93. The method of claim 91, wherein said predetermined angle is
based on a maximum phase shift between said first and second
polarization components due to a change adjacent to said
surface.
94. The method of claim 86, wherein said first light beam has
substantially circular polarization downstream of said interface.
Description
CROSS-REFERENCE
[0001] This application is a Continuation application which claims
the benefit of U.S. application Ser. No. 13/929,731, filed Jun. 27,
2013; which claims the benefit of U.S. application Ser. No.
11/735,900, filed on Apr. 16, 2007, now U.S. Pat. No. 8,488,120,
which is a continuation-in-part application of U.S. patent
application Ser. No. 11/379,026, filed Apr. 17, 2006, now U.S. Pat.
No. 7,233,396, which are all incorporated herein by reference in
their entirety.
FIELD ON THE INVENTION
[0002] This invention relates to affinity bio- and chemical sensors
and is based on measurement of the phase shift between two
orthogonal polarization components for quantitative analysis of
liquid and gaseous samples.
BACKGROUND OF THE INVENTION
[0003] Scientists and industry alike are continually seeking
methods to evaluate molecular interactions and eliminate the
uncertainty associated with utilizing labels to detect the location
of molecules of interest. Label-free technologies are crucial in
terms of addressing this issue, as these techniques allow
researchers to look at molecular systems without perturbing them
with extraneous chemistries that fundamentally change the dynamics
of interaction. The sensitivity of instruments designed to analyze
these molecular interactions is of paramount concern because often
molecules of interest are difficult and expensive to produce and/or
isolate, or are present in biological samples only at very low
concentrations. Compounding the issue of miniscule quantities are
the numerous variations of analytes, such as in drug development's
combinatorial chemistry libraries of which binding characteristics
are desired. It is desirable to develop sensors capable of
integration with high throughput screening methods. It must also be
sensitive enough to detect precious amounts of interesting
molecules, quickly and specifically.
SUMMARY OF THE INVENTION
[0004] A system comprising a light source, a sensor, an optical
retarder, a beam splitter, and a detector for determining the
optical properties of a sample material is disclosed. The system
comprises a light source for generating a first light beam having
first and second lightwaves, the first lightwave having a first
linear polarization and the second lightwave having a second linear
polarization, the first and second linear polarizations being
orthogonal to each other, and the first and second lightwaves being
in phase relative to each other. The first linear polarization
(p-polarization) being in the plane of incidence, and the second
linear polarization (s-polarization) being normal to the plane of
incidence. The intensity of the first lightwave and second
lightwave are set to a predetermined ratio. The system also
includes an optical retarder for providing a variable phase shift
between the first and second lightwaves by imposing a relative
delay between the first and the second lightwaves and a prism
interface for reflecting the first light beam from the sample
material under Total Internal Reflection (TIR) or Frustrated Total
Internal Reflection (FTR) conditions.
[0005] A polarizing beam splitter is used for splitting the first
light beam into a second light beam and a third light beam after
the first light beam is reflected from the optical interface, where
the second light beam and the third light beam comprise combined
projections of mutually orthogonal polarization components on the
main axes of the beam splitter. A signal processor measures the
intensity difference between the second and the third light beams
to calculate the phase difference induced by the sample material,
the signal processor receiving a first intensity measurement from a
first detector and a second intensity measurement from a second
detector where the first and second detectors measure the
intensities of the second and third light beams, respectively.
[0006] The light source for the system may comprise a coherent
light beam that is linearly polarized and with the linear
polarization rotated at a predetermined angle with respect to the
plane of the sensor surface. The incident angle is determined based
on maximum phase shift between the first and second lightwaves due
to a change on the sensor surface. The light source may be a gas
laser, a diode pumped solid state laser, an excimer lamp, a
vertical cavity surface emitting laser, a laser diode, or any light
source that will provide a linearly polarized coherent light having
a wavelength range of about 500-700 nanometers.
[0007] The polarizing beam splitter may be oriented such that the
intensity of the second and third lightbeams are substantially
equal or equal to one another. The polarizing beam splitter may be
oriented at about a 45 degree angle to the plane of the optical
interface.
[0008] The predetermined ratio of the intensities of the first and
second lightwaves is 13 where:
.beta. = I P 0 I P 1 = I P 0 I S 0 , ##EQU00001##
and where I.sup.0.sub.P and I.sup.I.sub.P are intensities of the
first lightwave before and after reflection from the sample
material, respectively, and I.sup.0.sub.S is the intensity of the
second lightwave.
[0009] The optical retarder in the system may be a Fresnel rhomb
prism or a right angle prism to provide a substantially 90 degree
phase shift between orthogonal polarization components.
[0010] The system may further comprise one or more optical
components located in the path of the first beam where the one or
more optical components convert substantially elliptical
polarization into substantially circular polarization before the
light beam is transmitted to the beam splitter.
[0011] The system comprises at least one sensor and may comprise at
least one SPR transducer. The sensor may contain sensing material
comprising biological molecules, such as antibodies, antigens,
oligonucleotides, proteins, enzymes, receptors, receptor ligands,
organic molecules, and catalysts. Alternatively sample material may
be applied to an array of transducers, each transducer containing
sensing material, with subsequent analysis by the sensor. The
sample material may comprise nucleic acid, proteins, polypeptides,
organic molecules, bacteria and viral particles.
[0012] The system of the invention is capable of detecting surface
refractive index change of at least 5.times.10.sup.-8 Refractive
Index Units. The system is capable of detecting amounts of sample
of at least 50 femtograms or at least 2,230,000 molecules of a 100
amino acid peptide, when associated with the molecularly-specific
surface.
[0013] In another aspect of the present invention a method is
provided of determining the optical properties of a sample
material. The method comprises steps of applying the sample
material to an optical interface of a transducer containing sensing
material, generating a first light beam having first and second
lightwaves, the first lightwave having a first linear polarization
and the second lightwave having a second linear polarization, the
first and second linear polarizations being orthogonal to each
other. A substantially 90 degree phase shift between the first and
second lightwaves, after the first light beam is reflected from the
sensor surface, is provided by imposing a relative delay between
the first and the second lightwaves. The first light beam reflects
from the sample material under TIR or FTR conditions, whereupon the
first light beam is split into a second light beam and a third
light beam after the providing and the reflecting steps, where the
second light beam and the third light beam comprise or consist of
combined projections of mutually orthogonal polarization components
of the first light beam. The intensity difference is measured
between the second and the third light beams to calculate the phase
difference induced by the sample material.
[0014] In yet another aspect of the invention, a method is provided
to detect the presence of analyte in a sample, comprising the steps
of applying the sample to an optical interface of a transducer
containing sensing material; reflecting a first light beam off the
optical interface, wherein the first light beam has a first and a
second lightwave; splitting the first light beam after it is
reflected from the optical interface into a second light beam and a
third light beam; and measuring the difference in intensities
between the second and the third light beams to calculate a phase
difference induced by the sample.
[0015] In the methods of the invention, the first light beam may be
linearly polarized and coherent. The first light beam may have
first and second lightwaves. The first lightwave may have a first
linear polarization and the second lightwave may have a second
linear polarization. The first and second linear polarizations are
orthogonal to each other. The first light beam, after being
reflected from the sensor surface, may be substantially
elliptically polarized and may be converted into substantially
circular polarized light prior to splitting the first light beam
into the second and third light beam. A 90 degree or substantially
90 degree phase shift is provided by reflecting the first light
beam from two reflection surfaces under total internal reflection
conditions, and/or by means of adjusting optical retarder in the
path of the first light beam.
[0016] In the methods of the invention, the predetermined ratio of
the intensities of the first and second lightwaves is .beta.
where:
.beta. = I P 0 I P 1 = I P 0 I S 0 , ##EQU00002##
and where I.sup.0.sub.P and I.sup.I.sub.P are intensities of the
first lightwave before and after reflection from the sample
material, respectively, and I.sup.0.sub.S is the intensity of the
second lightwave.
[0017] In some cases the intensity of the second and third light
beams may be equal or substantially equal. The second and the third
light beams may consist of combined projections of mutually
orthogonal polarization components on the main axes of a beam
splitter.
[0018] The systems herein can include sensing material immobilized
on the optical interface. Such material can comprise e.g.,
biological molecules, such as antibodies, antigens,
oligonucleotides, proteins, enzymes, receptors, receptor ligands,
organic molecules, and catalysts. The sample material may comprise
nucleic acid, proteins, polypeptides, organic molecules, bacteria
and viral particles. Sample material may be applied to one
transducer or to an array of transducers, each transducer
containing sensing material, with subsequent analysis by the
sensor.
[0019] The methods of the invention are capable of detecting
surface refractive index change of at least 5.times.10.sup.-8
Refractive Index Units (RIU) and of detecting amounts of sample of
at least 50 femtograms or at least 2,230,000 molecules of a 100
amino acid peptide, when associated with the molecularly-specific
surface. The analysis may be performed in the presence of a
reference sample, with or without analyte present, and with or
without analyte of known identity and amount present in the
reference sample.
INCORPORATION BY REFERENCE
[0020] All publications and patent applications mentioned in this
specification are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated to be incorporated by
reference.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0022] FIG. 1 illustrates the TIR sensor using the Fresnel rhomb
approach in accordance with an embodiment of the present
invention.
[0023] FIG. 2 shows an embodiment of the present invention using a
right angle prism configuration.
[0024] FIG. 3 shows a sensor array in accordance with an embodiment
of the invention.
[0025] FIG. 4 shows a bio-sensor array chip for sensing
applications that may operate in accordance with the invention.
[0026] FIG. 5 illustrates a typical response of the system as a
bio-affinity sensor.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Optical sensors based on the detection of analyte binding to
thin receptor films at the sensor surface have been studied
intensively. The use of an optical Total Internal Reflection (TIR)
configuration for measurement of index of refraction changes in the
evanescent field is common to interferometers, ellipsometers and
polariscopes. All these techniques can be applied to the
measurements of chemical or biological layered media, with the goal
of label free detection.
[0028] Internal reflection ellipsometry (IRE) may be used for
orientation of liquid crystals and absorption of solutes onto
substrate surfaces and for measurements of the refractive index of
liquids. Measurements were conducted in the total internal
reflection region of the incident angle that is not favorable for
thickness measurements owing to low sensitivity. In an effort to
enhance the sensitivity of such thickness measurements, the
implementation of the Surface Plasmon Resonance (SPR) effect has
been developed--a method falling under the more general Frustrated
Total Internal Reflection (FTR) approach. However, there remains a
need for highly sensitive devices for measurements of both
thickness and index of refraction changes in bio- and chemical
sensing devices.
[0029] Several methods have been employed to measure the spatial
reflection coefficients and overall intensity from the sensor
interface. These intensity-based techniques suffer from the
fluctuation of intensity in light sources and the relatively
small-reflected coefficient from the sensor surface. Higher
detection sensitivity is always desirable for improving sensing
performance. High sensitivity may be achieved utilizing other
factors. It has been found that a lightwave's phase can change much
more abruptly than the intensity when the refractive index or
thickness of a binding layer on the surface has been changed.
Several methods may be employed in sensors by measuring the phase
change from the sensor interface during SPR, even with the
capability of sensor array imaging. For example, a sensor based on
the combination of SPR and heterodyne interferometry with extremely
high sensitivity and low-noise was proposed.
[0030] During both TIR and FTR conditions the phase difference
between p- and s-polarized components of reflected beam experiences
a rapid shift whenever the optical properties of an adjacent medium
change, such as refractive index or thickness of the affinity
sensitive layer. Moreover measurement of the phase shift of the
p-polarized component of the incident beam yields significantly
higher sensitivity than SPR techniques that measure intensity
associated with incident angle change. Furthermore, the
phase-change method allows using both metal coated and optically
transparent transducers without a special metallic coating.
[0031] The method disclosed herein applies in a similar manner to
both TIR and FTR conditions in terms of measured parameters and
general configuration of the system. Utilizing the SPR phenomena in
sensing applications has been demonstrated in several different
configurations. A common approach uses the Kretschman
configuration. A coherent p-polarized optical wave is reflected
under TIR conditions on an interface between an optically dense
material, such as a glass prism, and a rarefied medium, which in
this case is the sample medium, whose index of refraction is lower
than the dense medium. The interface between the two media is
coated with a thin conductive metal film, which acts as an absorber
for the optical wave. When specific conditions dependent on the
light wave's angle of incidence, wavelength and the media's
refractive indices are met, the optical wave causes the metal's
surface plasmon electrons to oscillate at resonance, absorbing the
wave's energy in the metal film. During these resonance conditions,
variations in the sample's index of refraction will produce sharp
changes to the optical phase of the p-polarized component, while
the s-polarization phase remains relatively constant. At the
resonant conditions, most of the p-polarization light component in
contrast to TIR condition is absorbed in the metal film via the SPR
effect. This fact is exploited by intensity based SPR sensors and
ellipsometers, relating the conditions of the intensity minimum of
the reflected light to the optical configuration to thereby deduce
the sample's index of refraction or layer thickness.
[0032] The described approach uses a single light beam
configuration and monitors any changes in the layered media by
observing the intensity of orthogonal polarization components (i.e.
p- and s-polarizations, the p-polarization being in the plane of
incidence and the s-polarization being normal to the plane of
incidence), contained within this beam, reflected from the
interacting TIR/SPR surface, and where the beam's polarization
components experience intensity and phase shifts, the observation
of which is optimized through obtaining a substantially 90.degree.
phase shift between the polarizations in the steady state sensor
condition.
[0033] The method herein makes use of Polarization Based
Interferometry (PBI) coupled with Total Internal Reflection (TIR)
or Frustrated Total Internal Reflection (FTR) conditions to enable
a level of sensitivity previously unattainable by other competing
technologies. Interferometric measurement of two polarization
states (s-polarization and p-polarization) of the same beam in
combination with FTR elevates the sensitivity to surface refractive
index change to at least 5.times.10.sup.-8 Refractive Index Units
(RIU), which permits detection of at least 50 femtograms of sample,
or at least 2,230,000 molecules of a 100 amino acid peptide, when
associated with the molecularly-specific surface.
[0034] The present method is applied under either TIR or FTR
conditions. The phase difference between p- and s-polarization
components of incident beam experiences a rapid shift when
dielectric properties of the medium adjacent to the transducer
surface change. The phase of the p-polarization component changes
much more abruptly than the intensity when the refractive index or
thickness of a binding layer on the surface has been changed. This
difference allows the use of a single substantially linearly
polarized beam tilted under a certain adjustable angle for
sensitive detection of refractive index change, on or in close
vicinity to, the transducer surface. The highest sensitivity is
achieved when the two components are in phase-quadrature or
90.degree. out of phase. The combination of obtaining orthogonal
polarization components with a polarizing beam splitter and
differential signal processing scheme provides direct, highly
sensitive measurement of small phase shifts and multiplex array
imaging detection.
[0035] The present invention provides a method for evaluation of
multiple complementary chemistries in an array format that enables
high throughput screening at high sensitivity level. Potential uses
for such a device are infectious disease screening in blood and
detection of virus or bacterial particles in air, pharmacokinetic
research and primary binding studies for drug development
applications in addition to personalized medicine, particularly for
cancer, detection of bio- and chemical warfare agents/hazards, and
quality control for industrial processes.
[0036] Some examples of infectious disease screening may include;
identification of whole bacteria, whole virus or viral particles,
whole infectious parasites or other lifecycle congeners thereof;
identification of antibodies raised against specific bacterial or
viral strains; excreted/secreted antigens or nucleic acid markers
from infectious agents; or protein biomarkers of disease either
from the host or disease causing agent. Infectious disease
screening could be use for either diagnostic purposes or for blood
supply safety monitoring. Personalized medicine may use
identification of antibodies, proteins, peptides, nucleic acids,
and small organic molecules which arise from the disease state of
interest. In pharmaceutical research and development, the present
invention may be used in screening of nucleic acids, antibodies,
proteins, peptides and synthetic organic molecules to identify
active molecules suitable for development as a drug candidate;
detection of drug candidates in biological samples throughout
preclinical and clinical development; and safety monitoring of
subjects in clinical and community settings, for detection and
quantification.
[0037] Other potential uses may include air and water quality
monitoring. Monitoring can be for particulates, pollutants, toxins,
industrial waste products, waste biologics, or inorganic health
hazards.
[0038] FIG. 1 shows an optical TIR sensor 10 with an embodiment of
the proposed invention. Sensor 10 comprises a light source 100, a
half-wave plate 200, a Fresnel rhomb 300, a polarized beam splitter
400, two photo detectors 500 and 600, and a slide containing a
specific sensor material 700 which interacts with the sample
800.
[0039] Light source 100 can be any light source that provides
substantially monochromatic coherent radiation, such as a laser.
Preferably, the light source is a gas laser, a diode pumped solid
state laser, an excimer lamp, vertical cavity surface emitting
laser or a laser diode. Preferably, light source 100 is any light
source that will provide substantially monochromatic coherent
radiation in the 500-700 nanometer wavelength range. Light source
100 is used to generate a substantially linearly polarized light
beam S. Half-wave plate 200 is used to rotate the polarization of
light beam S and to provide orientations of the polarization of the
beam S at 45 degrees off the vertical axis. As a result signal S1
contains both s- and p-polarization components where the phase
difference between the s- and p-polarization is zero degrees.
[0040] Fresnel rhomb 300 is used to introduce a substantially 90
degree phase difference between the s- and p-polarization
components of signal S1. Other known methods of introducing a 90
degree phase shift between the s- and p-polarization components of
signal S1 are also contemplated by the present invention. A slide
700 containing a specific sensor material is placed on one of the
Fresnel rhomb surfaces, either R1 or R2 or both. Preferably, slide
700 is made of a material that is substantially transparent to the
wavelengths of light source 100. Preferably, slide 700 is made of a
material that has a higher index of refraction than the sample.
Preferably, slide 700 is made of glass, plastic, silicon or
ceramic. The sensor material in slide 700 interacts with the sample
800. This interaction produces a phase shift between the s- and
p-polarization components when signal S1 reflects from the
rhomb/slide boundary. Slide 700 and sample 800 are optically
coupled to the rhomb 300 to ensure minimal losses and to maintain
total internal reflection conditions.
[0041] Alternatively, slide 700 and prism 300 are part of a single
assembly thereby eliminating the need to use index matching fluid.
In one embodiment, the angle of incidence of the light beam from
light source 100 maybe varied around the optimum TIR/FTR angle for
a particular application. The incident angle is determined based on
the maximum phase shift between the first and second lightwaves due
to a change on the sensor surface. The exact value of the incidence
angle may be determined from the condition of yielding maximum
sensitivity of the sensor and approximate linearity of the
instrument response within a desired dynamic range even if the
operating conditions change, i.e. new chip, variation in refractive
of the metal or bio-coating, etc. In this embodiment, the signal
that changes proportionally with the incident angle will be
modulated at that frequency. Then the jittering frequency will be
filtered out and will not affect the measured DC signal.
Preferably, the frequency is significantly higher than the
characteristic time of the measured process.
[0042] Polarized beam splitter 400 is used to combine the
projections of the s- and p-polarized components of signal S2. Beam
splitter 400 can be any beam splitter that combines the orthogonal
polarization components. In some embodiments, beam splitter 400 is
a cube beam splitter. The separated projections of the s- and
p-polarization components are detected by detectors 500 and 600.
Detectors 500 and 600 are any photo-detectors that accept an
optical signal and generate an electrical signal containing the
same information as the optical signal, e.g. a photodiode (PD) or a
charge coupled device (CCD).
[0043] The electrical signals generated by detectors 500 and 600
are transmitted to a signal processing unit 900. The signal
processing unit 900 reads the difference between the powers on the
two detectors. This difference is directly proportional to the
phase shift between the s- and p-polarization components. The phase
shift between the s- and p-polarization components is indicative of
changes in the optical properties, e.g. index of refraction or
thickness of sensor material on slide 700.
[0044] FIG. 2 shows a sensor system 30 in accordance with an
embodiment of the present invention. Sensor system 30 comprises a
linear polarized light source 314, a half-wave plate 320, a right
angle prism 310, a polarized beam splitter 340, and detectors 350
and 360. Light source 314 can be any light source that provides
substantially monochromatic coherent radiation, such as a laser. In
some embodiments, the light source is a gas laser, a diode pumped
solid state laser, an excimer lamp, vertical cavity surface
emitting laser or a laser diode. In some embodiments, light source
100 is any light source that will provide substantially
monochromatic coherent radiation in the 532-680 nanometer
wavelength range. Preferably, light source 314 is a tunable laser
that provides a range of wavelengths. Light source 314 is used to
generate a substantially linearly polarized optical beam I.
Half-wave plate 320 is used to rotate the polarization of optical
signal I by 45 degrees to generate signal I1. Half-wave plate 320
can also be used to adjust the relative optical power between the
s- and p-polarizations at the TIR/SPR sensing surface, for signal
optimization in accordance with equation (13) below. Beam I1
contains both s- and p-polarization components with a zero degree
phase difference between them.
[0045] Beam I1 is sent to prism 310 where it attains elliptical
polarization. The sensor system 30 also includes a host glass
substrate 370, a metal film 390 with a bio-affinity coating
applied, which interacts with the sample 380. Preferably, metal
film 390 is a gold film. The right angle prism 310 may be rotated
to accommodate for liquid or gas operation, or a different
wavelength light source. Beam I1 experiences TIR condition at
surface 318 of the prism, bounces off the second TIR surface 322
within the prism and exits the prism as beam I2. Beam I2 is
elliptically polarized. Waveplates 311, 312 are used to achieve a
beam I3 having substantially circular polarization (substantially
90 degree phase shift). In some embodiments, one of the wave plates
is fixed with its fast axis at 45 degrees with respect to the plane
of incidence. Optionally, wave plate 316 is used to obtain a
substantially 90 degree phase difference. Both of these methods can
be applied when the range of index of refraction for measured
sample buffers is too broad and substantially 90 degree phase can
not be achieved by the TIR reflections in the prism alone.
Polarizing beam splitter 340 combines the projections of s- and
p-polarization components of beam I3. Beam splitter 340 can be any
beam splitter that combines the orthogonal polarization components
of a single beam. Preferably beam splitter 340 is a cube beam
splitter.
[0046] The combined projections of the s- and p-polarization
components are detected by their respective detectors 350 and 360.
Detectors 350 and 360 are any photo-detectors that accept an
optical signal and generate an electrical signal containing the
same information as the optical signal such as a photodiode (PD) or
a charge coupled device (CCD). The electrical signals generated by
detectors 350 and 360 are transmitted to a signal processing unit
330. The signal processing unit 330 reads the difference between
the powers on the two detectors. This difference is directly
proportional to the phase shift between the s- and p-polarization
components. The phase shift between the s- and p-polarization
components is indicative of changes in the optical properties, e.g.
index of refraction or thickness of sensor material on metal film
390.
[0047] Optionally prism 310 may be rotated to accommodate different
types of samples. During operation, if the sample reference leads
to a significant change in the initial critical SPR angle, optimum
operation might require a rotation of the prism 310. Optionally,
the prism 310 may be dynamically rotated during a measurement. The
shown configuration lets the input and output light beams from the
prism 310 remain parallel even if the prism is rotated, thus
additional dynamic alignment during operation is avoided.
[0048] The method of the present invention exploits the fact that
the polarization state or the relative phase between s- and
p-polarizations and the intensity of each individual polarization
component, changes when the ratio of indices of refraction at an
optical interface changes under total internal reflection (TIR) or
frustrated total internal reflection (FTR) conditions. The method
of the present invention seeks to extract the maximum possible
difference between the changing s- and p-polarization components by
imposing a substantially 90 degree phase shift between these
components.
[0049] For pure TIR conditions the optical phase shift (.delta.)
between the p- and s-polarization components is expressed as:
.delta. = .delta. p - .delta. s = 2 tan - 1 [ cos .theta. i sin 2
.theta. i - n 2 2 / n 1 2 sin 2 .theta. i ] ( 1 ) ##EQU00003##
[0050] Where .theta..sub.i is the light beam incident angle on the
specific TIR/FTR interface, n.sub.2 and n.sub.1, the refractive
indices of the sample medium and the prism 310, respectively. The
incident angle may be fixed for a given instrument, with a built in
static gain compensation system for simple systems, or variable, so
as to accommodate for a range of buffer solution and SPR
conditions, in order to achieve the highest possible sensitivity in
more complex sensor systems.
[0051] For SPR, the reflection coefficients of the s- and
p-polarizations can be written:
r.sub.P=|r.sub.P|e.sup.i.delta.p,r.sub.S=|r.sub.S|e.sup.i.delta.s
(2)
[0052] And the phase difference between the p- and s-polarization
components is:
.delta.=.delta..sub.P-.delta..sub.S (3)
[0053] which can be found from the overall complex reflection
relation (2).
[0054] In general the substantially linear polarized beam incident
on the sensing surface turns into elliptically polarized light when
exiting the prism 310. The beam's electromagnetic vector is
described by:
E.sub.S=a.sub.1cos(.tau.+.delta..sub.S) (4)
E.sub.P=a.sub.2cos(.tau.+.delta..sub.P) (5)
[0055] Where .delta..sub.1 and .delta..sub.2 are the phase shifts
for the s- and p-polarizations respectively, and:
r=.omega.t-{right arrow over (k)}{right arrow over (r)} (6)
[0056] If two detectors D.sub.1 and D.sub.2 are located
orthogonally and turned by angle .PSI. in respect to the plane of
incidence, one can obtain the difference in power between them as
such:
D.sub.2-D.sub.1=.DELTA.I=1/2(a.sub.1.sup.2-a.sub.2.sup.2)cos
2.PSI.+a.sub.1a.sub.2 sin 2.PSI. cos .delta. (7)
[0057] If detectors are located at angle such as
tan 2 .PSI. * = a 2 2 - a 1 2 2 a 1 a 2 cos .delta. , then
##EQU00004## D 1 - D 2 = 0. ##EQU00004.2##
[0058] Assuming now that the following assumptions hold:
a 2 = a 20 + .DELTA. a 2 , .DELTA. a 2 a 20 << 1 ( 8 )
.delta. = .delta. 0 + .DELTA. .delta. , .DELTA..delta. .delta. 0
<< 1 ( 9 ) ##EQU00005##
[0059] Here a.sub.20 and .delta..sub.0 refer to initial or steady
state conditions. From this, it follows:
.DELTA.I|.sub.0=1/2(a.sub.1.sup.2-a.sub.20.sup.2)cos
2.PSI.+a.sub.1a.sub.20 sin 2.PSI. cos .delta..sub.0 (10)
.DELTA.I|.sub.a.sub.2,.delta..apprxeq.1/2 cos
2.PSI.(a.sub.1.sup.2-a.sub.20.sup.2+2a.sub.20.DELTA.a.sub.2)+a.sub.1(a.su-
b.20+.DELTA.a.sub.2)sin 2.PSI. cos(.delta..sub.0+.DELTA..delta.)
(11)
[0060] The change in the differential intensity due to a change on
the transducer surface therefore is:
R=[.DELTA.a.sub.2(a.sub.1 sin 2.PSI. cos .delta..sub.0-a.sub.20 cos
2.PSI.]-{a.sub.1a.sub.20 sin 2.PSI. sin .delta..sub.0 sin
.DELTA..delta.} (12)
In equation (12) the first bracket refers to the "intensity"
contribution, whereas the second bracket corresponds to the "phase"
contribution of the response R. During TIR the intensity does not
change and only the phase shifts. However both intensity and the
phase change during SPR event. Nevertheless it was shown that the
intensity contribution can be neglected, thus the sensor response
is simplified to:
R=-a.sub.1a.sub.20 sin 2.PSI. sin .delta..sub.0 sin .DELTA..delta.
(13)
[0061] A key concept of the mentioned approach is to maximize the
response to phase changes.
[0062] The phase shift contribution is maximized when these
conditions are met:
sin 2 .PSI. .fwdarw. 1 .PSI. .fwdarw. .pi. 4 ( 14 ) sin .delta. 0
.fwdarw. 1 .delta. 0 .fwdarw. .pi. 2 ( 15 ) a 20 = a 1 ( 16 )
##EQU00006##
The condition (15) refers to the phase quadrature or substantially
90.degree. phase shift, as previously described. Condition (14)
indicates that the detector should be located 45.degree. off the
plane of incidence. The conditions (14) and (16) can be also
satisfied by rotating the half-wave plate located before the
sensing prism to compensate for absorption of p-polarized component
of the beam at the metal surface during SPR event. The rotation
angle .eta. can be given as:
tan .eta. = .beta. = I P 0 I P 1 = I P 0 I S 0 , ( 17 )
##EQU00007##
where I.sup.0.sub.P and I.sup.I.sub.P are intensity of
p-polarization component before and after reflection from sensed
surface, and I.sup.0.sub.S is the intensity of the s-polarized
component.
[0063] Finally, the maximum response that corresponds to circular
polarization and is presented as:
R=-a.sub.1.sup.2 sin .DELTA..delta.=I.sub.0 sin .DELTA..delta.
(18)
[0064] To obtain the desired phase difference of substantially
90.degree. various methods of phase retardation can be used in the
beam path. Specific examples to achieve the desired phase shift
could be implemented by varying the following parameters: the prism
310 index of refraction, the index ratio on any non-sensor TIR/FTR
interfaces within the prism, the incident angle .theta..sub.i, and
the number of TIR/FTR reflections within the prism.
[0065] For instance, a variable wave plate is a z-cut birefringent
material, such as crystal quartz. The wave plate is designed such
that the index of refraction varies with the tilting angle .alpha.,
with respect to the optical axis, in a range between n.sub.0 and
n.sub.e, where:
n.sub.e= {square root over (n.sub.0.sup.2 cos.sup.2
.alpha.+n.sub.e.sup.2 sin.sup.2 .alpha.)}
[0066] The wave plate has an index of refraction of n.sub.0 when
positioned perpendicular to the beam, .alpha.=0, and a value of
n.sub.e when aligned parallel to the beam. Therefore one can adjust
the phase to be substantially 90.degree. at the polarizing beam
splitter 340 independent of what phase difference comes from the
SPR at the metal film 390.
[0067] In addition, should the polarization not be completely
circular, but a compressed vertical or horizontal ellipse (still
substantially 90.degree. phase shift between s and p), a half wave
plate may be placed in front of the prism to balance the power of
the two polarization components after the interaction at the
sensing surface, creating the desired circular polarization.
[0068] Yet another way to achieve circular polarization is by the
use of a liquid crystal polarization rotator that gives an easy way
to control the polarization angle in response to a signal reading.
For example, an applied voltage can be adjusted with a feed-back
loop to provide a close to zero differential reading between the
two polarization components.
[0069] Thus, various methods can be used to obtain the maximum
output signal and phase difference quadrature. For instance, one
can use two or more reflection surfaces to provide a substantially
90.degree. phase shift between the two polarization components.
This can be achieved by design of a special prism with proper
refractive index that provides TIR conditions upon every
reflection. With the SPR approach, a specially designed prism with
two or more reflections can be used to provide a substantially
90.degree. phase shift between s- and p-polarization. If the range
of index of refraction for any intended sample buffer is too broad,
substantially 90.degree. phase shift may not be achieved without
the use of the variable wave plate 316. In addition, two .lamda./4
wave plates (311, 312) may be placed after the prism 310, to
convert the elliptically polarized beam to a circularly polarized
beam, thus obtaining a substantially 90.degree. phase shift.
[0070] FIG. 3 illustrates an embodiment of the present invention
where the sample 413 is an array of sample transducers such as the
AlphaSniffer virus chip 950 shown in FIG. 4. A chip 950 is
illustrated in FIG. 4. The virus chip 950 in FIG. 4 can be on any
substrate that is substantially transparent to light source 100
(FIG. 1) or 314 (FIG. 2). In one embodiment, chip 950 is on a glass
or an SF11 substrate. Chip 950 has properties similar to other
Lab-on-Chip or Bio-Chip approaches. In one embodiment, chip 950 can
have open "affinity sensing features" on the surface exposed to
analyzed liquid or the fluidics channel can be built as a part of
the chip assembly. The fluid path can be a single channel or
multi-channel. It's preferable to have a carrier stream (buffer).
Preferably, the sample is degassed prior to delivery to the chip to
avoid gas bubble formation on or in close vicinity of the chip
surface
[0071] FIG. 3 shows an affinity bio- and chemical sensor 40 for
processing multiple sample transducers 380 (See FIG. 4) using the
method of the present invention. Sensor 40 comprises a transducer
array 413 that comprises an array of transducer elements 380 and a
corresponding metal film layer 390 (not shown) for each element to
makeup the transducer array 413. The beam reflects off this array
and retains the phase and intensity information for each element
380. A beam splitter 440 is used to combine the projections of the
s- and p-polarization components which are then detected by
detectors 415 and 414. Detectors 414 and 415 comprise a
photodetector array or a CCD camera. The detectors 414 and 415 are
used to obtain an "image difference" using a differential signal
analysis tool using techniques similar to the ones described above.
The "image difference" contains phase information for each of the
transducer elements 380 in array 413.
[0072] FIG. 5 shows test results for a sample analyzed using a
device embodying the present inventions. Three consecutive
injections of 120 .mu.L of avidin solution in buffer at 0.3 mg/mL
concentration are injected over the sensing surface coated with
biotinated BSA. Delta Response is the difference between response
resulting from binding avidin to the surface of the transducer and
initial response of the system while the buffer is flowing over the
transducer. Incremental increase of the Delta Response shows that
the surface of the transducer was not saturated from the first
injection of avidin sample and there was additional binding during
consequent injections. The method also allows investigation of the
rate of the reaction by varying the volume and concentration of
injected analyte and the time of exposure of the sample to the
surface of the transducer.
[0073] The systems and methods disclosed herein can be used for
evaluation of multiple complementary chemistries in a sample or
plurality of samples. The samples are preferably fluidic in nature
(e.g., liquid, gas, etc.). Examples of a fluid sample that can be
obtained from an animal include, but are not limited to, whole
blood, serum, sweat, tears, ear flow, sputum, lymph, bone marrow
suspension, urine, saliva, semen, vaginal flow, cerebrospinal
fluid, brain fluid, ascites, milk, secretions of the respiratory,
intestinal and genitourinary tracts, and amniotic fluid.
[0074] The samples can be analyzed to detect and/or identify
particular analyte(s) in the sample based the analyte(s)'
complementary chemistries. Examples of complementary chemistries
include, but are not limited to, antibody-antigen interactions,
including sandwich antibody interactions, receptor-ligand
interactions, including small molecules, peptides, and proteins,
interaction with both natural and synthetic receptors, nucleic
acids interrelations and other interactions between compositions,
for example non-receptor mediated protein/protein, peptide, small
organic molecule or inorganic molecule interactions.
[0075] For example, for analysis of antibody-antigen interactions,
transducers can be composed of biofilms (e.g., sensor materials)
where the biofilm is either composed with or coated with an
antibody or an antigen for detection of complementary chemistry in
any biological fluid such as blood, plasma, saliva, urine, bile,
etc. or other media mix. The above can be used to detect, for
example, a polypeptide or protein in a sample by using a transducer
composed of biofilm(s) with an antibody that specifically binds the
polypeptide or protein of interest. Useful applications of such
embodiments include infectious disease diagnostics including blood
supply safety, medical diagnostics, personalized medicine such as
characterization and diagnosis of cancer, bio/chemical hazard or
bio/chemical weapons detection, including detection of protein- and
small molecule-based toxins and poisons, and immunological
research. For any of these broader applications, the instrument
would be employed for detection of viral or disease-related
antigens, host immunoglobulins, viral particles and/or
bacteria.
[0076] Compared to enzyme linked immunosorbent assays, (ELISA) the
proposed system offers distinct benefits that include a much
quicker time to results since incubation with additional reporter
antibodies and activation enzymes is eliminated, and real time
monitoring, as well as significant reduction of complexity in terms
of assay process. Reduction of background signal due to
non-specific binding of complex mixtures such as blood, a simple
step that is similar to the blocking process used for ELISA studies
is proposed--such that the complex mixture absent the analyte of
interest would be passed over the transducer, non-specific binding
measured, and then complex mixture containing the analyte of
interest is added, resulting in signal specific to the analyte of
interest. Additionally, information related to the rates of
association and dissociation of the analytes of interest to the
binding molecules on the biosensor can be derived.
[0077] In some embodiments, the systems and methods herein can be
used to detect receptor-ligand interactions. In such embodiments, a
receptor or ligand is bound to the transducer surface and the
binding of the ligand to it assayed. Normally such interactions are
monitored using a reporter system such as colorimetric/fluorescent
or radioactive labels. The methods and systems herein allow for
real-time, multiplexed combinatorial chemistry screens for drug
development, pharmacokinetics, biochemical kinetic studies for
association and dissociation constants, quality control of
industrial processes where undesirable products or side reactions
may take place, monitoring completion of chemical reactions,
catalysis, ion-implantation and basic chemistry research. Where
precious metals are used in catalytic processes such as
hydrogenation on platinum, real time analysis and evaluation of the
process may be possible via direct integration of the catalytic
surface as the transducer. Kinetic studies in both drug discovery
and biological research can be performed by immobilizing a
biological molecule of interest, an enzyme, for example, and permit
the measurement of association and dissociation or processing rate
of a particular small molecule or putative biological target.
Synthetic receptors may also be immobilized, i.e., for example,
modified porphyrins, cyclodextrins, amongst others, to detect small
molecules capable of selective binding.
[0078] In some embodiments, the systems and methods herein detect
and/or monitor nucleic acid (e.g., DNA) hybridization with
exquisite sensitivity. This can be used, for example, to detect
hybridization of a target nucleic acid sequence to a probe on an
array. Analysis of nucleic acid hybridization also permits
determination of viral load, and eliminates the need for a nucleic
acid amplification step if sufficient numbers (hundreds of
thousands) of molecules are present to register signal, as opposed
to tens of billions that other systems demand. Again, the systems
and methods herein allow for the real-time detection of such
analytes (e.g., target nucleic acid sequences) in a sample.
[0079] Other forms of molecular target analysis that can be
performed by the present invention include detection of virus
particles or other particles of biological origin in building air
supply systems, chemical and biological warfare agents, water and
food quality, monitoring chemical contaminants in feed and waste
streams or environmental life support systems and power plant
emissions. Whole bacteria may also be detected as well, in either
fluid or gaseous samples. Thus, the present invention relates to
infectious disease screening in blood and detection of virus or
bacterial particles in air, pharmacokinetic research and primary
binding studies for drug development applications in addition to
personalized medicine, particularly for cancer, detection of bio-
and chemical warfare agents/hazards, and quality control for
industrial processes.
[0080] In any of the embodiments herein, the devices can be
configured to enable high throughput screening using an array of
transducers having a different probe or sample at discrete sites.
For instance, chips with from ten to 1000 features could be
developed to analyze small nuclear polymorphisms (SNPs), or
specific hereditary markers for prenatal screening, or to determine
a cancer's susceptibility to various drug regimens, or evaluate the
genetic nature of psychological disease, or analyze blood samples
from numerous patients for infectious disease such as hepatitis C
or HIV. Analysis rate depends in part on the number of discrete
sites on a chip and also on the sample volume. In some embodiments,
a chip is designed with more than but not limited to 10, 100, 1,000
or 10,000 discrete sites. In some embodiments, an apparatus herein
analyzes up to but not limited to 120 .mu.L of a sample in 1-2
minutes or up to but not limited to 500 .mu.L of a sample in 2-5
minutes. Discrete sites can have samples containing, e.g., blood or
other bodily fluid, water, gaseous material, etc. Each of the
discrete sites can have an address such that data collected from
one sample from a unique site can be associated with a particular
sample (e.g., a sample collected from a patient X).
[0081] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now be apparent to those skilled in the art without departing from
the invention. It should be understood that various alternatives to
the embodiments of the invention described herein may be employed
in practicing the invention. It is intended that the following
claims define the scope of the invention and that methods and
structures within the scope of these claims and their equivalents
be covered thereby.
* * * * *