U.S. patent application number 11/805774 was filed with the patent office on 2008-11-27 for sensor apparatus and method using optical interferometry.
Invention is credited to Lucien P. Ghislain.
Application Number | 20080291456 11/805774 |
Document ID | / |
Family ID | 40072091 |
Filed Date | 2008-11-27 |
United States Patent
Application |
20080291456 |
Kind Code |
A1 |
Ghislain; Lucien P. |
November 27, 2008 |
Sensor apparatus and method using optical interferometry
Abstract
A sensor apparatus and method includes a sensor head with at
least two surfaces separated by a gap. One surface is mechanically
fixed, a second surface is free to move and deflections of the
second surface relative to the first surface are monitored by
optical interferometry. In one embodiment, an optical fiber is used
to direct light from a light source to the sensor and collect light
reflected by the sensor. In alternate embodiments the sensor
apparatus includes integrated optical elements, free-space optics,
and direct laser-diode sensing. In operation, interaction of
molecules or other objects in the sample with the second surface is
detected as a change in amplitude and/or phase of deflection the
second surface in response to an applied driving signal. A layer of
binding molecules may be immobilized on the second surface and this
surface exposed to a sample. The invention includes a method for
detecting an analyte in a sample, including detecting the presence
of analyte, the amount of analyte or the rate of association and/or
dissociation of the analyte with a binding partner.
Inventors: |
Ghislain; Lucien P.;
(Millbrae, CA) |
Correspondence
Address: |
LUKE P. GHISLAIN
1380 MILLBRAE AVE.
MILLBRAE
CA
94030
US
|
Family ID: |
40072091 |
Appl. No.: |
11/805774 |
Filed: |
May 24, 2007 |
Current U.S.
Class: |
356/450 ;
73/649 |
Current CPC
Class: |
G01N 2291/02827
20130101; G01N 2291/02466 20130101; G01N 33/54373 20130101; G01N
29/022 20130101; G01N 29/032 20130101; G01N 2291/0427 20130101;
G01N 2291/02881 20130101 |
Class at
Publication: |
356/450 ;
73/649 |
International
Class: |
G01B 9/02 20060101
G01B009/02; G01N 29/00 20060101 G01N029/00 |
Claims
1. A device comprising: a support including a first surface; a
second surface positioned proximate to the first surface, the
second surface free to deflect in response to a driving force; a
driving means capable of generating a driving force to deflect the
second surface; a detector operable to monitor deflections of the
second surface; a sample including at least one analyte, the sample
exposed to the second surface and the analyte able to interact with
the second surface; where interaction of the analyte with the
second surface is detected as a change in the deflection of the
second surface.
2. The device of claim 1 where the driving means is an external
transducer.
3. The device of claim where there the driving means is integrated
into the device.
4. The device of claim 1 where the driving force is an
electrostatic force.
5. The device of claim 1 where the driving means includes a
transducer capable of generating acoustic or ultrasonic pressure
waves.
6. The device of claim 1 where the second surface includes a film
including a magnetized material disposed on at least one side of
said second surface.
6a. The device of claim 1 where the second surface is electrically
conductive.
7. The device of claim 1 where the driving means comprises: a
signal source; a magnetic field generator generating a magnetic
field directed toward the second surface and driven by the signal
source.
7a. The device of claim 1 where the driving means comprises: a
signal source generating a current in the second surface: a
magnetic field generator, where the magnetic field generates a
force on the current in the second surface causing a deflection of
the second surface.
8. The device of claim 1 including a synchronous detector with the
signal from the detector monitoring second surface deflections as
input and the signal from the driving means as reference.
9. The device of claim 1 where the detector is an optical
interferometer capable of monitoring deflections of the second
surface relative to the first surface.
10. The device of claim 1 where both first and second surfaces are
capable of reflecting light.
11. The device of claim 1 where the second surface is a thin
circular membrane held in tension
12. The device of claim 1 where the detector monitors changes in
the amplitude of deflection of the second surface.
13. The device of claim 1 where the detector monitors changes in
the phase of the deflection of the second surface relative to the
phase of the driving force.
14. The device of claim 1 where the driving means operates in a
pulsed mode and the dissipation of the oscillation of the second
surface is monitored.
15. The device of claim 1 where the second surface includes a layer
of analyte binding molecules.
16. The device of claim 1 operating to detect specific molecular
binding of at least one analyze to the second surface.
17. The device of claim 1 where the sample is a fluid.
18. The device of claim 1 where specific analyte binding to the
second surface changes the detected deflection of the second
surface.
19. A method of detecting analyte binding comprising; positioning a
first surface proximate a second surface, the first surface
including a exposing the second surface to a sample including at
least one analyte so that the analyte is free to internet with the
second surface. applying a driving force to deflect the second
surface, monitoring the deflection of the second surface in
response to the driving force, detecting the interaction of the
analyte with the second surface as a change in the deflection of
the second surface.
20. A device comprising: a support including a first surface; a
second surface positioned proximate to the first surface; a
solenoid coil proximate the second surface; a signal generator; an
optical interferometer monitoring deflections of the second
surface; a synchronous detector with the optical interferometer
signal as input and the signal generator as reference; a sample
including at least one analyte; where the first and second surfaces
can reflect light, where the second surface includes a magnetic
film, where the signal generator drives the solenoid coil to
generate a magnetic field, where the magnetic field generates a
deflection of the second surface, where the second surface is free
to deflect in response to a driving force, where the sample is
exposed to second surface, where the at least one analyte can
specifically bind to the second surface, where the optical
interferometer measures the deflection of the second surface
relative to the first surface, where the synchronous detector
monitors the amplitude of the second surface deflection, where the
synchronous detector monitors the phase of the second surface
deflection relative to the phase of the signal generator, where the
binding of the analyte to the second surface is detected as a
change in the deflection of the second surface and monitored by the
synchronous detector.
20a. A device comprising: a support including a first surface; a
second surface positioned proximate to the first surface; a
permanent magnet proximate the second surface; a signal generator;
an optical interferometer monitoring deflections of the second
surface; a synchronous detector with the optical interferometer
signal as input and the signal generator as reference; a sample
including at least one analyte; where the first and second surfaces
can reflect light, where the second surface is free to deflect in
response to a driving force, where the second surface is
electrically conductive, where the sample is exposed to second
surface, where the at least one analyte can specifically bind to
the second surface, where the signal generator generates an
electric current in the second surface, where the optical
interferometer measures the deflection of the second surface
relative to the first surface, where the synchronous detector
monitors the amplitude of the second surface deflection, where the
synchronous detector monitors the phase of the second surface
deflection relative to the phase of the signal generator, where the
binding of an analyte to the second surface is detected as a change
in the deflection of the second surface and monitored by the
synchronous detector.
Description
FIELD OF THE INVENTION
[0001] The invention relates to an apparatus and method for
detecting the presence, amount, or rate of binding of one or more
analytes in a sample, and in particular, to apparatus and method
using optical interferometry to monitor displacement of a sensor
element.
BACKGROUND OF THE INVENTION
[0002] High sensitivity, real-time, label-free monitoring of
binding to a surface in a liquid, in a gaseous environment, or in a
vacuum has a wide range of applications, including thin film
thickness monitoring during deposition, biological applications for
molecular, viral, bacterial and cellular detection, surface
science, surfactant research, drug research and discovery,
electrochemistry (including plating and etching) and in situ
monitoring (e.g. oil condition).
[0003] Diagnostic tests based on a binding event between members of
binding pair are widely used in medicine, agriculture, food
analysis and research. These tests are designed to detect the
presence, amount, rate of binding of a wide variety analytes.
Typical binding pairs include antibody-antigen, receptor-ligand,
DNA or RNA hybridizing pairs.
[0004] In a solid-phase assay molecules are immobilized on a solid
surface, the solid surface is exposed to a sample under conditions
that promote binding, binding occurs in a defined detection zone,
and binding events may be detected by a variety of direct and
indirect methods. Methods of direct detection include a change in
mass, viscosity, elasticity, dissipation, electric charge or
potential, surface stress, reflectivity, thickness, color.
[0005] Methods of indirect detection include the use of a
chromophore or fluorescent label, and radiolabels. Further, binding
can be detected after it occurs by a secondary fluorescent-labeled
anti-analyte antibody.
[0006] Prior art patent U.S. Pat. No. 5,804,453, "Fiber optic
direct-sensing bioprobe using a phase-tracking approach" issued to
Chen; Duan-Jun, and related U.S. Application No. 20050254062
"Fiber-optic assay apparatus based on phase-shift interferometry"
issued to H. Tan et al. discloses an optical fiber interferometer
assay device that is designed for direct detection of binding to an
optical fiber end surface. Detection is based on a change in
thickness at the optical fiber end surface due to molecular
binding. The change in thickness changes an optical interference
signal due to the phase shift between light reflected from two
layers on the optical fiber end surface: a first layer and a second
layer that is directly exposed to the sample. The prior art optical
fiber interferometer analyzes the phase shift between the first and
second layers using an optical spectrometer operating with visible
light in the range 450-700 nm. The phase shift due to binding is
detected by changes in the reflected light spectrum over a range of
wavelengths, specifically, changes in the peaks and valleys of the
reflected light optical spectrum.
[0007] One limitation of the prior art optical fiber interferometer
is the relatively low sensitivity. The measured phase shift depends
on a change in refractive index and the change in refractive index
due to binding of the analyte molecules is extremely small. As a
result a large number of molecules are needed to produce a
detectable signal. In addition, small changes in the positions of
peaks and valleys in the optical spectrum are difficult to detect,
and the signal may be weak and buried in noise.
[0008] Prior art patent U.S. Pat. No. 6,436,647, "Method for
detecting chemical interactions between naturally occurring
biological analyte molecules that are non identical binding
partners" issued to Quate, C. F. et al. discloses a method of using
cantilevers as sensors for detecting chemical interactions between
naturally occurring bio-polymers which are non-identical binding
partners. The method is useful whether the reactions occur through
electrostatic forces or other forces. Induced stress, heat, or
change in mass is detected where a binding partner is placed on a
cantilever for possible reaction with analyte molecules (i.e., a
non-identical binding partner). The method is particularly useful
in determining DNA hybridization but may be useful in detecting
interaction in any chemical assay.
[0009] Also, prior art patent U.S. Pat. No. 6,289,717,
"Micromechanical antibody sensor", issued to Thundat et al.
discloses a sensor apparatus using a microcantilevered spring
element having a coating of a detector molecule such as an antibody
or antigen. A sample containing a target molecule or substrate is
provided to the coating. The spring element bends in response to
the stress induced by the binding which occurs between the detector
and target molecules. Deflections of the cantilever are detected by
a variety of detection techniques. The microcantilever may be
approximately 1 to 200 .mu.m long, approximately 1 to 50 .mu.m
wide, and approximately 0.3 to 3.0 .mu.m thick. Sensitivity for
detection of deflections is in the range of 0.01 nanometers.
[0010] One disadvantage of cantilever deflection based sensors is
relatively low sensitivity due to the fact that a detectable
cantilever deflection is caused by the cumulative effect of the
binding of a large number of molecules to a cantilever surface.
This can take a significant amount of time, and therefore the
sensor response may be relatively slow. Further, the slow response
leaves the sensor susceptible to a variety of noise sources (drift,
thermal noise). In addition, the cantilever is a relatively rigid
free-standing element, rather like a swimming pool diving board and
as a result, the surface mass density of typical cantilever sensors
is much greater than the surface mass density of the molecules
bound to its surface.
[0011] In addition, the cantilever sensor has an inconvenient form
factor. Cantilever sensors may require an optical alignment each
time they are replaced. This is generally inconvenient and
particularly inconvenient in the case of multiple cantilever
sensors forming an array. Further, cantilever sensors may be too
expensive for operation as a single-use disposable, and re-use over
many cycles may be difficult to achieve.
[0012] U.S. Pat. No. 5,807,758, "Chemical and biological sensor
using an ultra-sensitive force transducer", issued to Lee et al.
discloses a method and apparatus for detecting a target species.
The target molecule may be in liquid phase (in solution) or (for
some embodiments of the invention) in vapor phase. A sensor
according to the present invention monitors whether a target
species has selectively bound to groups on the cantilever surface
by monitoring the displacement of the cantilever, and hence the
force acting on the cantilever. This force acting on the cantilever
arises from the force acting on a structure that moves in electric
or magnetic field, and that may be selectively bound to the
cantilever. In the case of target species having a sufficiently
large net electric charge or dipole moment, the target species
itself may serve as the structure that moves in an electric field.
More typically however, separate modified structures, such as
modified magnetic beads or modified beads having a net charge or a
dipole moment, will, when selectively bound to the cantilever,
exert a force on the cantilever that relates to the presence of the
target species.
[0013] This is a cantilever-based approach that has the additional
disadvantage of requiring a label and thus it provides only an
indirect measure of analyte binding.
[0014] U.S. Application No. 20040096357, "Composite sensor
membrane", issued to Majumdar et al. discloses a sensor including a
membrane to deflect in response to a change in surface stress,
where a layer on the membrane is to couple one or more probe
molecules with the membrane. The membrane may deflect when a target
molecule reacts with one or more probe molecules.
[0015] This is a membrane sensor that operates on the same
principle the cantilever sensors to detect analyte binding by
changes in surface stress. It has the disadvantages of the
cantilever sensors as previously described. Specifically, the
sensitivity may be relatively low because it requires a large
number of binding events. The sensor response may be relatively
slow and is generally susceptible to noise and drift. In addition,
the membrane is a relatively rigid free-standing element and the
surface mass density of the membrane may be much greater than the
surface mass density of the molecules bound to its surface.
Further, the membrane sensor may be too expensive to be a
single-use disposable, and re-use over many cycles may be difficult
to achieve.
[0016] A related sensor, the quartz crystal microbalance (QCM) is
an electro acoustic method suitable for mass and viscoelastic
analysis of adsorbed protein layers at the solid/water interface. A
typical QCM sensor consists of a megahertz piezoelectric quartz
crystal sandwiched between two gold electrodes. The crystal can be
brought to resonant oscillation, and shear motions by means of A/C
current between the electrodes. Since the resonant frequency (f)
can be determined with very high precision, usually less than 1 Hz,
the adsorbed mass at the QCM-surface can be detected, or
"balanced", down to a few ng/cm2. It has also been shown that there
is linear relation between the adsorbed rigid mass and the change
in f, in an ideal air/solid situation.
[0017] U.S. Pat. No. 6,006,589 "Piezoelectric crystal microbalance
device", issued to Rodahl et al. discloses a device and a process
for measuring resonant frequency and dissipation factor of a
piezoelectric resonator. After exciting the resonator to
oscillation, the driving power to the oscillator is turned off
after the decay of the oscillation of the resonator is recorded and
used to give a measure of at least one of the resonators
properties, such as dissipation factor, changes in the dissipation
factor, resonant frequency and changes in the resonant frequency.
The invention allows these measurements to be performed at either
the fundamental resonant frequency or one (or more) of the
overtones. The device and the process disclosed herein may be used
in a variety of applications such as, for example, measurement of
phase transitions in thin films, the detection of adsorption of
biomolecules, and measurements of the viscoelastic properties of
thin films.
[0018] The crystal microbalance sensor typically vibrates in a
shear mode with an amplitude of about 1 nm at a fundamental
resonant frequency given by:
fres.about.1/d
where d is the thickness of the quartz plate. For example, if d is
0.17 mm, the resonance frequency is approximately 10 MHz. The
quartz sensor starts to oscillate if an AC electric field with a
frequency centered close to the fundamental resonant frequency of
the sensor is applied perpendicularly to its surfaces. Usually,
electrodes on each side of the sensor plate are deposited by
evaporation and are subsequently contacted to an external AC field
generator (for example to a signal generator, or to an oscillator
drive circuit, or the like). Under favorable conditions this
arrangement is capable of sensing mass changes smaller than 1
ng/cm{circumflex over (0)}2.
[0019] Ideally the mass changes at the sensor electrode(s) induce a
shift in the resonance frequency of the sensor, proportional to the
mass changes:
Delta M=-C Delta fres
where C, the proportionality constant, depends on the thickness of
the quartz plate.
[0020] This relation is valid provided that the mass is attached
rigidly to the electrode and follows the oscillatory motion of the
sensor without dissipative losses. The relation may fail when the
added mass is viscous or is not rigidly attached to the
electrode(s) and can thus suffer elastic or plastic deformation(s)
during oscillations. The relation between added mass and the shift
of the resonant frequency then becomes more complex. The latter
situation arises when for example a water droplet is deposited onto
an electrode of the quartz sensor.
[0021] The Sauerbrey equation describes the linear relation between
frequency changes and changes in mass for thin films adsorbing to
the crystal microbalance sensor surface. It gives a good estimation
of film thickness, as long as the dissipation is relatively low.
When the dissipation value reaches above 1.times.10-6 per 5 Hz, the
film is too soft to function as a fully coupled oscillator. A
calculated thickness value will hence be somewhat less than the
true value.
[0022] Proteins at the water/QCM surface interface can also be
quantified with resonance frequency determination, but adsorbed
protein layers also have some degree of structural flexibility or
viscoelasticity, that are invisible to a simple resonance frequency
determination. Viscoelasticity can, however, be visualized by
measuring the energy loss, or dissipation (D) of the shear movement
of the crystal in water. A new principle of measuring D is to drive
the crystal with A/C current at the resonant f followed by
disconnection and analysis of the resulting damped sinusoidal
curve. This development of pulse assisted discrimination of
resonance frequency and dissipation makes QCM analysis of adsorbed
protein layers very simple and gives unique information about the
hydrodynamic conductivity of the adsorbed protein layers and
surrounding water. Very small structural and orientation changes of
an adsorbed protein layer, including chemical cross-linking, may be
monitored with high accuracy.
[0023] One disadvantage of this approach is the piezoelectric
crystal sensor size--typically 10-30 mm diameter and 0.1-0.5 mm
thickness. Also, the entire sensor element including piezoelectric
crystal and electrodes is typically thrown out after each use. In
some cases, the sensor element can be reused a number of times, but
this requires careful cleaning. In addition, sensor size limits
scalability to an array-sensing format for multiple analytes.
[0024] A preferred embodiment of the sensor apparatus of the
invention includes an optical-fiber based interferometer to measure
small displacements. One such interferometer is disclosed in U.S.
Pat. No. 5,017,010, "High sensitivity position sensor and method",
issued to Mamin et al. This is a highly sensitive apparatus for
sensing the position of a movable member comprises an optical
directional coupler providing four external ports. Light from a
short coherence length diode laser is injected into the first port.
The coupler serves as a beam splitter to direct one portion of the
injected light to the member via the second port and a single mode
optical fiber. Part of this one portion is reflected concurrently
from the member and from the adjacent polished coating at the end
face of said fiber back into said fiber and optically coupled via
the third port to a photodetector to provide a signal whose
amplitude is indicative of the position of the member, based upon
the relative phase of said concurrent reflections. The other
portion of the injected light is optically coupled to and via the
fourth port to another photodetector for providing, as a reference,
a signal proportional to the intensity of the injected light. These
two signals are supplied to a subtractive circuit for providing an
output in which power fluctuations of the laser are minimized.
[0025] Another embodiment of the invention uses a laser-diode based
interferometer for direct sensing of small displacements. One such
laser-diode based interferoineter is disclosed in U.S. Pat. No.
5,189,906, "Compact atomic force microscope", issued to Sarid et
al. This is an atomic force microscope using a laser diode and
optical interference of light reflected back into the laser to
measure the vertical position of a sensing tip wherein the sensing
tip can be either off the sample surface and vibrated where changes
in the amplitude of vibration near the natural frequency of the
cantilever are used as a measure of changes of electric or magnetic
force on the sensing tip; or, the sensing tip can be placed on the
sample surface with no vibration to measure directly the profile of
the sample surface.
SUMMARY OF THE INVENTION
[0026] The invention includes an apparatus for high sensitivity,
real-time, label-free monitoring of binding to a surface in a
liquid, in a gaseous environment, or in a vacuum. The invention
also includes an apparatus for detecting an analyte in a sample,
including detecting the presence of analyte, the amount of analyte
or the rate of association and/or dissociation of the analyte with
a binding partner.
[0027] In a preferred embodiment the apparatus includes: a sensor
head with at least two surfaces separated by a gap. The first
surface may be mechanically fixed, for example, a reflective layer
coated on an optical fiber end surface. The second surface may be
free to move, for example a membrane or diaphragm capable of
reflecting light and supported over the circumference but otherwise
free to move. A layer of binding molecules immobilized on the
second surface is exposed to a sample. A light source generating
light that is directed to the reflecting surfaces and a light
detector monitoring the light reflected by the reflecting
surfaces.
[0028] The apparatus may also include a driving signal generator
capable of generating pressure waves in the sample with selectable
frequency and amplitude ranging from acoustic to ultrasonic
frequencies (from 1 Hz to 1 GHz). The driving signal generator may
be formed from any of a wide variety of actuating elements,
including: a piezoelectric devices (for example PZT, PVDF, quartz),
magnetically driven elements, electrically driven elements (using
electrostatic forces or an electric current), thermally driven
elements (for example, a resistive heating element), optically
driven elements. The pressure wave generator may be mounted
separately from the sensor head or may be included as a component
of the sensor head.
[0029] Alternatively, the second surface may be driven directly by
use of actuating elements on or near the sensor head similar to
those described above and including: a piezo-electric devices (for
example PZT, PVDF, quartz), magnetically driven elements,
electrically driven elements (using electrostatic forces or an
electric current), thermally driven elements (for example, a
resistive heating element), optically driven elements. For example,
a surface coated with a magnetic film respond to an applied
external magnetic field. Electrostatic forces can deflect the
second surface in response to an applied voltage when electrodes
are included on or near the two sensor surfaces. In addition, the
spacer layer sandwiched by the first and second surfaces may be
formed from a piezo-electric material with suitable electrodes. A
voltage applied to the electrodes can drive a displacement of the
second surface.
[0030] In one embodiment, the second surface moves in response to
pressure waves in the sample and this motion is monitored as a
change in the signal from the light detector. More specifically,
the light source generates light that illuminates the first and
second surfaces and the light detector operates to monitor light
reflected by the two surfaces. Interference between the two
reflected light beams provides a measure of the relative position
of the two surfaces. When the second surface moves there is a shift
in the phase of the reflected light relative to the light reflected
by the first surface. This phase shift changes the light intensity
monitored by the light detector. When the sensor head is exposed to
the sample, molecules or other objects in the sample can bind to
the second surface, and this is detectable as a change in the
motion of the second surface in response to an applied pressure
wave or driving signal.
[0031] The sensor may detect the binding of a wide variety of
analytes in a fluid sample, including: antibodies, antigen
molecules, protein molecules for detecting binding partners,
protein molecules for detecting protein complexes, DNA strands
capable of hybridization. The sensor may also detect binding of
particles, virus capsids, cells and larger objects of interest. In
addition the sensor may detect a variety of analyte in a vapor
(gas-phase, or both liquid and vapor phase) sample, including:
volatile molecules, compounds and contaminants. Further, the sensor
may operate in vacuum to detect the deposition a sample on the
sensor surface or collision (with or without binding) of a sample
with the sensor surface.
[0032] The sensor can also operate without the use of an external
driving signal generator. For example, movement of the second
surface could be generated by a sample including motile cells (for
example, sperm and bateria). Further, movement could be generated
by sample particles, molecules, cellular components or even whole
cells and larger objects carried by fluid flow in or around the
sensor head.
[0033] In one particular design the two reflecting surfaces are
separated by an air gap. The sensor head may be exposed to a liquid
sample with one side of the second surface exposed to the liquid
and with an air-gap maintained on the second side in the space
between the first and second surfaces.
[0034] In another design the two reflecting surfaces are separated
by a gap that is directly exposed to the sample (vapor or liquid),
and one or both sides of the second surface may be exposed to the
sample.
[0035] The second surface may be a diaphragm or membrane, a ribbon
or strip, a perforated membrane having a pattern of holes, a
reflecting surface supported by one or more flexible arms
[0036] The overall size of the first and second surfaces can be
selected for optimum sensitivity. For example, in the case of a
membrane or diaphragm the diameter may be in the range 10 cm (10-1
meters) down to 10 nm (10-8 meters), with smaller sizes tending to
provide higher sensitivity and faster response times.
[0037] The light source can include an optical fiber for directing
light to the sensor head and the sensor apparatus further includes
an optical coupling for directing reflected light to the light
detector.
[0038] In a first embodiment, the sensor head is fixedly mounted to
an optical fiber with the first reflecting surface in direct
contact with the optical fiber end surface.
[0039] In a second embodiment, the sensor head is removably
attached to the optical fiber and another optical element is
sandwiched between the optical fiber end face and the sensor
head.
[0040] In an alternate embodiment, free-space optical elements
(lenses, mirrors, beam-splitters, filters and similar components)
direct light from a light source to the sensor, collect light
reflected by the sensor and direct reflected light to the light
detector.
[0041] In a further embodiment, integrated optical elements
(waveguide, coupler, Mach-Zehnder) direct light from a light source
to the sensor and collect light reflected by the sensor, and direct
reflected light to the light detector.
[0042] In a still further embodiment, the sensor is mounted
adjacent to a laser diode. Light emitted by the laser diode is
incident on the sensor and some of the light reflected by the
sensor returns to the laser diode. The monitor photodiode included
in the laser-diode housing is used to monitor the light reflected
by the sensor.
[0043] For detecting multiple analytes the sensor apparatus can
include of an array of discrete analyte-binding regions, each
region can be effective to bind a different analyte. In one
embodiment, the optical fiber includes a plurality of individual
fibers each aligned with one of the analyte-binding regions, and
the detector includes a plurality of detection areas the optical
coupling functions to couple the detection areas with the optical
fibers.
[0044] In another aspect, the invention includes a method for
detecting the presence or amount of an analyte in a sample. The
method involves exposing the sensor head to the sample. Allowing
the analyte in the sample to react and bind with the analyte
binding molecules immobilized on the second surface. Analyte
binding is measured by detecting a change in the amplitude and
phase of the motion of the second surface in response to an applied
driving signal. The motion is detected by monitoring the optical
interference between the reflected light beams from the first and
second surfaces to produce a sensor signal.
[0045] In one method of detection, the driving frequency is
adjusted to be near a resonance frequency of the sensor, and the
sensor oscillation amplitude is measured. Analyte binding to the
sensor changes the total oscillating mass and thus also changes the
sensor resonance frequency. For operation in fluid, all the
oscillating mass including trapped and bound water will be
measured.
[0046] In another method of detection, the driving frequency is
adjusted to be near a resonance frequency of the sensor, and the
phase of the sensor oscillation relative to the driving signal is
measured at nearly constant amplitude. Analyte binding to the
sensor results in a phase shift that can be monitored with high
sensitivity.
[0047] In another method of detection, the driving frequency is
adjusted to be near a resonance frequency of the sensor and the
dissipation is measured by periodically turning off (pulsing) the
driving signal and monitoring the decay of the sensor oscillation.
Binding to the sensor surface damps the oscillation and changes the
dissipation, D, defined as the sum of all energy losses in the
system per oscillation cycle. It is also defined as 1/Q, i.e. the
energy dissipated per oscillation, divided by the total energy
stored in system. Rigid structures tend to give low dissipation,
while soft structures give higher dissipation especially if there
is a lot of coupled water.
[0048] In another method of detection, the driving frequency is
adjusted to be near multiple resonance frequencies (harmonics,
overtones) of the sensor. If the sample is rigidly coupled to the
sensor surface, the dissipation is low, and each of the resonance
frequencies will show similar response. If the sample is soft and
does not fully couple to the sensor oscillation, the dissipation is
higher, and each of the resonance frequencies may show a different
response.
[0049] The detecting step can include directing light from an
optical fiber onto the two reflecting surfaces and directing
reflected light from the two surfaces onto a light detector via an
optical coupling. The detector can be a photodiode where detecting
includes monitoring changes in light intensity due to optical
interferences between the two reflected light beams.
[0050] Where the method is used for measuring the rate of
association of analyte to the second surface, the sensor signal can
be continuously monitored as analyte binding occurs, until a
maximum is reached.
[0051] Where the method is used for measuring the rate of
dissociation of analyte from the second surface, the reacting steps
can include exposing the second surface in a dissociation
environment and continuously monitoring changes in the sensor
signal until a minimum is reached.
[0052] Where the method is used for measuring the amount of analyte
present in the sample, the detection is carried out continuously
over a period of time sufficient to measure changes in the sensor
signal at a plurality of time points.
[0053] Where the method is used to measure one or more of a
plurality of analytes in a sample, the sensor head is composed of
an array of discrete analyte-binding regions, each region being
effective to bind a different analyte. The detection step includes
monitoring the change in motion of each region of the second
surface resulting from binding of analyte to the analyte-binding
molecules.
[0054] Objects of the Present Invention Include:
[0055] To provide a sensor that operates with speed and
simplicity.
[0056] To provide a sensor that significantly improves sensitivity
and accuracy.
[0057] To provide a sensor that can operate frequencies well above
DC.
[0058] To provide a sensor that can monitor both amplitude and
phase.
[0059] To provide a sensor that can operate without the use of
labels for real-time and direct detection of binding.
[0060] To provide a sensor that can be made very small.
[0061] To provide a sensor capable of measuring a small sample
volume.
[0062] To provide a low-cost disposable sensor head format.
[0063] To provide a sensor that easily scales to a multiplexed
array-sensing format for measuring multiple analytes
simultaneously.
[0064] To provide a sensor suitable for operation in a vacuum,
gaseous, or liquid environment.
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] FIG. 1. is a schematic diagram an embodiment of the
apparatus using optical fibers.
[0066] FIG. 2. shows one embodiment of the sensor head
[0067] FIG. 3. illustrates the optical interference signal.
[0068] FIG. 4. is a schematic of another embodiment of the sensor
head.
[0069] FIG. 5. is a schematic diagram an alternate embodiment of
the apparatus using free-space optics.
[0070] FIG. 6. is a schematic of another embodiment of the sensor
head compatible with free-space optical apparatus.
[0071] FIG. 7. is a schematic diagram an alternate embodiment of
the apparatus using laser diode sensor.
[0072] FIG. 8. is a schematic of a multiplexed embodiment of the
sensor head suitable for multiple analytes.
DETAILED DESCRIPTION OF THE INVENTION
[0073] FIG. 1A is a schematic of the sensor apparatus 100 according
to the invention and includes a light source 110, sensor head 120,
light detector 130 for detecting optical interference signals from
the light waves reflected by the sensor head. An optical coupling
140 assembly directs light to the sensor head and back to the light
detector. In one embodiment, the light source, coupler, sensor head
and light detector are connected using optical fiber 150, 152, 154.
In another embodiment, the light source, coupler, sensor head and
light detector are connected using integrated optical waveguides.
In the preferred embodiment, the optical coupling assembly 140
includes a first optical waveguide or fiber 150, 152 connecting the
light source to the sensor head and a second optical waveguide or
fiber 154 connecting the sensor head to the light detector. An
optical coupler, well known in the art, optically couples the first
and second waveguides or fibers. The light source 110, optical
coupler 140, and light detector 130 components are all commercially
available. Signal processor 132 generates a driving signal that is
directed to transducer 122. In one embodiment transducer 122 is a
solenoid coil that can generate deflections of sensor head
components that include magnetic material. In another embodiment
transducer 122 is a piezoelectric element capable of producing
acoustic or ultrasonic waves in a sample.
[0074] In another embodiment, shown in FIG. 1B transducer 122 is
integrated into the sensor head 120 and sensor head components may
be driven directly by use of actuating elements on or near the
sensor head similar to those described above and including: a
piezo-electric devices (for example PZT, PVDF, quartz),
magnetically driven elements, electrically driven elements (using
electrostatic forces or an electric current), thermally driven
elements (for example, a resistive heating element), optically
driven elements. Electrostatic forces can deflect the second
surface in response to an applied voltage when electrodes are
included on or near the two sensor surfaces. In addition, the
spacer layer sandwiched by the first and second surfaces may be
formed from a piezo-electric material with suitable electrodes.
[0075] FIG. 2 shows a sensor head assembly 120 according to one
embodiment of the invention. The sensor head assembly includes: a
first surface 160 that is capable of reflecting light, a second
surface 170 that is capable of reflecting light and a gap 180
separating the first and second surfaces. The first and second
surfaces 160, 170 may have thickness in the range 0.1 nanometer
10-10 meter) to 1 centimeter (10-2 meter). In a preferred
embodiment the second surface 170 is a membrane or diaphragm with
diameter in the range 10 nanometers (10-8 meters) to 10 centimeters
(10-1 meters). The gap 180 may be an air gap and the gap height is
preferably set according to the wavelength of the light source to
be N lambda/4, where N is an integer, for the maximum sensitivity
to deflections of the second surface 170.
[0076] Analyte-binding molecules 190 may be immobilized on the
second surface 170 such that, when the sensor head 120 is exposed
to the sample, analyte molecules 200 specifically bind to the
second surface 170 with high affinity. A specific binding reaction
is by definition a saturable reaction, usually reversible, that can
be competed by an excess of one of the reactants. Specific binding
reactions are characterized by a complementarity of shape, charge
and other binding determinants as between the participants in the
specific binding reaction. The analyte and anti-analyte molecules
may be members of a binding pair, examples include: antibodies,
DNA, RNA, protein, small and large molecules, cells, receptors and
their binding targets, toxins.
[0077] The first surface 160 may be simply the bare end-face of an
optical fiber. In this case the reflectivity is determined by the
refractive index of the glass forming the fiber and the air or
fluid forming the gap. Alternately the first surface 160 may
include a single or multilayer coating to control the optical
reflectivity. For example, a layer of a higher refractive index
material (for example, Ta2O5) or a multilayer dielectric thin film
stack (alternating SiO2 and Ta2O5 as is well known in the art) may
be designed to produce the desired reflectivity. Further, the first
surface 160 may include a thin metal film (for example, gold) with
the thickness of the film selected to produce the desired
reflectivity. The first surface 160 functions to reflect a portion
of the light arriving from the light source 110 and is preferably
mounted on a rigid substrate.
[0078] The second surface 170 may also include a single layer or
multilayer thin film stack, for example a dielectric such as SiO2,
Ta2O5, a polymer layer or layers, or a metal thin film or metal
foil, similar to the first surface 160. The second surface 170 may
also be partially or fully reflective in order to reflect some or
all of the light arriving from the light source 110. The second
surface 170 is supported near the first surface 160 by a spacer
layer 180 that forms the gap but it is otherwise free to move in
response to an applied force or pressure. The gap distance is in
the range 1 nm (10-9 meters) -1 cm (10-2 meters) and selected
according to the wavelength of illumination in order to optimize
sensitivity. The thickness of the second surface 170 may be
selected according to the desired sensitivity, with sensitivity
increasing as the thickness decreases. In a preferred embodiment
the second surface 170 is supported by the spacer layer 180 to form
a membrane or diaphragm. The membrane or diaphragm may be held in
tension to control the resonance frequency and optimize
sensitivity. In the case of a liquid sample, the membrane may be
exposed to the sample on only one side. One the second side there
is a gap, preferably and air-gap, formed by the spacer layer 180
and the first surface 160.
[0079] In addition, the first surface, spacer layer, and second
surface may include polymer materials. For example, the spacer
layer may be formed from a piezo-electric film (for example, PVDF,
PZT, quartz). In addition, the first surface may include a polymer
layer to provide a flexible substrate for the spacer layer and
second surface.
[0080] In addition to the membrane or diaphragm there are a variety
of alternate configurations for the second surface. For example the
second surface may be formed by: [0081] a ribbon: a reflecting
surface supported at two ends. [0082] a trampoline: a reflecting
surface supported by three or more arms. [0083] a porous element:
for example a membrane, ribbon, trampoline having an array of
holes.
[0084] In the preferred embodiment, as analyte molecules 200 bind
to their binding partners 190 on the second surface 170, the added
material changes the response of the second surface 170 to an
applied force or pressure. In the special case where the second
surface forms a membrane with the following parameters:
TABLE-US-00001 radius r (meters) surface mass density Ma
(kg/m{circumflex over ( )}2) membrane surface tension T (N/m)
transverse wave velocity Vt (m/s)
[0085] Then Vt=SQRT(T/Ma) and the lowest resonance frequency
is:
.omega.res=2.40483 Vt/r (Hz)=2.40483/r SQRT(T/Ma) (Hz)
where the numerical factor 2.40483 is the first root of the zero-th
order Bessel function J0(x) and the entire membrane oscillates up
and own in this lowest order standing wave.
[0086] As an example, if we first consider a gold membrane in air
with radius 5 um, thickness 0.1 um.
[0087] The surface mass density determined from the volume density
and thickness: [0088] Mass density 19320 kg/m{circumflex over
(0)}3.times.0.1 um=1.932.times.10-3 kg/m{circumflex over (0)}2
[0089] Membrane surface tension is .about.0.1 N/m [0090] Then the
fundamental resonance frequency fres 550 KHz.
[0091] If the noise floor is approximately 0.01 Hz then the
smallest detectable change in mass density is given by the
relation:
.DELTA.Ma/Ma=2 .DELTA.fres/fres,
and the smallest detectable change in mass density is approximately
10 picogram/cm{circumflex over (0)}2=10-11 g/cm{circumflex over
(0)}2. This is just one example and even higher sensitivities may
be achieved by further optimization of the sensor parameters. It is
also possible to operate the sensor at higher harmonic or overtone
frequencies to obtain additional information about analyte binding.
This analysis is for the case of a membrane in air and does not
take into account the effects of a liquid. Generally, the presence
of a liquid will introduce viscous damping, tending to lower the
resonance frequency.
[0092] On the typical length scales of the sensor apparatus working
with a liquid sample, viscous forces dominate inertial forces. That
is, in the preferred embodiment, the Reynolds number is low and
fluid flows will be laminar (and usually not turbulent).
[0093] Conventional immobilization chemistries may be used to
attach the analyte-binding layer 190 to the second surface 170. The
analyte-binding layer 190 can be either a monolayer or a multilayer
matrix. A siloxane technique is available for attachment to glass
surfaces and a variety of techniques are available for attachment
to gold surfaces.
[0094] As shown in FIG. 3, the measurement of the presence,
concentration and/or binding rate of analyte to the sensor head 120
is enabled by the optical interference of the light waves reflected
by the two surfaces 160 and 170 in the sensor head. The operation
of the sensor in the preferred embodiment may be understood by
assuming that there are two reflected light waves from the first
and second surfaces 160, 170. The first surface 160 generates a
reflected wave with electric field E1, where the electric field is
a vector having both amplitude and direction, in this case we are
primarily concerned with the amplitude and phase. The second
surface 170 generates a reflected wave with electric field E2. The
interference of the two reflected light waves generates a new wave
with electric field:
E=E1+E2.
[0095] The light detector 130 measures the light intensity given
by:
Idetector=|Edetector|{circumflex over (0)}2|E1+E2|{circumflex over
(0)}2=I1+I2+2*I1*I2cos(2*pi*DELTA/Lambda)
[0096] Where DELTA is the optical path difference between the two
surfaces 160, 170 and lambda is the wavelength of the light from
the light source. The gap between the two surfaces is GAP=DELTA/2
n, where n is the refractive index of the material filling the gap.
In general, the electric fields E1 and E2 will have different
amplitudes, but in the special case where the amplitudes are the
same |E1|=|E2|=|E| the analysis is easier.
[0097] In this case, the maximum light intensity is:
Imax.about.|2E|{circumflex over (0)}2=4I,
[0098] This maximum light intensity occurs when the optical path
difference is:
DELTA=N Lambda, where N is an integer.
[0099] Also in this case, the minimum light intensity is:
Imin=|E|E|{circumflex over (0)}2=0
[0100] This minimum light intensity occurs when the optical path
difference is:
DELTA=N Lambda/2.
and when the optical path difference DELTA=(2N+1) Lambda/4, the
light intensity is:
Iquadrature2I
[0101] At this intensity, called the quadrature point, the light
intensity is in the middle of its range.
[0102] For example, in the special case where N=1 and air fills the
gap (n=1.0) and the wavelength Lambda=1000 nanometers (10-6
meters)
Imax occurs when GAP=500 nm
Iquadrature occurs when GAP=375 nm
Imin occurs when GAP=250 nm
[0103] So the detected light intensity varies from minimum to
maximum as the gap between the two reflecting surfaces varies by a
distance equal to 1/4 of the wavelength of the light source or, in
this case, 250 nm.
[0104] As described in more detail below, mechanical motion,
deflection or oscillation of the second surface 170 may be driven
by a variety of forces including: viscous, elastic, acoustic,
ultrasonic, inertial, thermal, electrostatic, magnetic,
piezo-electric and others.
[0105] In one embodiment, the second surface 170 is exposed to an
applied force or pressure generated by an external source. For
example, a piezo-electric element may be mounted in such a way that
it is exposed to the sample and driven by a driving signal to
generate acoustic or ultrasonic pressure waves in the sample, as is
well known in the prior art.
[0106] In another embodiment, the first or second surfaces 160, 170
may include a layer of magnetic material. An externally controlled
magnetic field, for example a solenoid coil, may be used to apply a
force to the magnetic layer, thus displacing the first surface
relative to the second surface.
[0107] In one implementation, as described by Lindsay in U.S. Pat.
No. 5,513,518, a thin film or particles of magnetic material 172 is
applied to the second surface 170 with the direction of the
magnetic moment M along the perpendicular to the plane of the
surface. A small solenoid 122 is placed near second surface 170 so
as to generate a magnetic field B that is predominantly
perpendicular to the magnetic moment of the magnetic film.
Construction of a suitable solenoid and choices of magnetic
materials is known to those of ordinary skill in the art. For the
purpose of modulating the deflection of the second surface 170, a
signal voltage source 134 is used to drive the solenoid 122. The
second surface deflection is detected by optical detector 130 and
delivered to a synchronous signal processor 132 which receives the
signal voltage 134 as a reference. The synchronous detector is used
to monitor both the amplitude and phase of the second surface
deflection, an adjustable time constant for integration of the
signal may be used to improve the signal to noise ratio.
[0108] An important step in the preparation of second surface 170
is the formation of a controlled magnetic moment. One method for
doing this is to place the surface in a strong magnetizing field.
In another method, a thin film of Cobalt is evaporated onto the
surface; such a film is relatively easy to magnetize in the plane
of the film.
[0109] In an alternate embodiment the sensor is driven directly by
use conductive elements, or electrodes, on or near the first and
second surfaces so that a voltage applied to the electrodes
generates an attractive force, by capacitive coupling, tending to
pull the first and second surfaces together as is well known in the
prior art.
[0110] In a further embodiment, the first or second surfaces 160,
170, the spacer layer 180 or one or more the mounting elements
shown in the figures, may include a piezoelectric element having
suitable electrodes. A driving signal voltage applied to these
electrodes generates a change in the shape or size of the
piezo-electric element generating a pressure wave and displacing
the first surface relative to the second surface.
[0111] Still further, the first or second surfaces 160, 170 may
include a resistive layer having suitable electrodes. A driving
signal voltage applied to these electrodes causes resistive heating
and leads to a change in shape or size of the resistive layer or
adjacent layers, thus displacing the first surface relative to the
second surface.
[0112] Still further, the first or second surfaces 160, 170 may
include electrodes suitable to carry electric current. An ambient
magnetic field (for example, the earths magnetic field) or an
externally controlled magnetic field, for example a solenoid coil,
may be used to apply a force to the current-carrying electrodes,
thus displacing the first surface relative to the second
surface.
[0113] Still further, the first or second surfaces 160, 170 may
include one or more layers of material that change shape or size in
response to temperature changes. It is then possible to displace
the first surface relative to the second surface by controlling the
temperature of the sample near the sensor head. For example, if the
second surface includes two layers of dissimilar metals, a
temperature change of the sample near the sensor head will cause a
displacement of the second surface relative to the first surface
due to the well known bimetallic effect.
[0114] In one method of operation, the sensor head 120 is exposed
to a sample that includes an analyte and the sensor head 120 is
also exposed to a drive signal from signal generator 134, of fixed
frequency and fixed amplitude. The frequency and amplitude of the
pressure wave are selected to optimize the response of the sensor
head 120. The second surface 170 moves in response to the applied
drive signal and this movement is detected as a change in the
optical interference signal at the light detector. Analyte binding
to the second surface changes the response of the second surface
170 to the applied drive signal, for example by changing the
fundamental resonant frequency of the second surface 170 to produce
a change in the amplitude of the second surface response.
[0115] In another method of operation, the sensor head 120 is
exposed to a sample that includes an analyte and the sensor head
120 exposed to a driving signal of known frequency, amplitude and
phase. The frequency, amplitude and phase of the driving signal are
selected to optimize the response of the sensor head 120, and may
be fixed or variable. The second surface 170 moves in response to
the applied driving signal and this movement is detected as a
change in the optical interference signal at the light detector.
Analyte binding to the second surface changes the response of the
second surface 170 to the applied driving signal and the phase of
this response can be compared to the phase of the driving wave
while the frequency and amplitude of the response are nearly
constant.
[0116] In an alternate method, the sensor head 120 is exposed to a
sample that includes an analyte and is also exposed to a drive
signal of fixed amplitude but variable frequency. The frequency can
be swept over a range anywhere from 1 Hz to 1 GHz, the frequency
can be set at a number of discrete frequencies or the frequency can
be swept continuously over a defined frequency range and then jump
to another defined frequency range, according to the optimum
response of the sensor head 120. The second surface 170 moves in
response to the applied drive signal and this movement is detected
as a change in the optical interference signal at the light
detector. The measured amplitude of the motion of the second
surface in response to the swept-frequency pressure wave is used to
generate an amplitude response spectrum. Analyte binding to the
second surface 170 changes the measured amplitude response
spectrum, for example by changing the fundamental resonant
frequency and also changing the harmonics or overtones.
[0117] In an alternate method, the sensor head 120 is exposed to a
sample that includes an analyte and is also exposed to a pressure
wave of variable amplitude and variable frequency. The frequency
can be swept over a range anywhere from 1 Hz to 1 GHz, the
frequency can be set at a number of discrete frequencies or the
frequency can be swept continuously over a defined frequency range
and then jump to another defined frequency range, and the amplitude
can be according to the optimum response of the sensor head 120.
The second surface 170 moves in response to the applied drive
signal and this movement is detected as a change in the optical
interference signal at the light detector. The measured amplitude
of the motion of the second surface in response to the
swept-frequency drive signal is used to generate an amplitude
response spectrum that can be normalized to the known driving
amplitude. Analyte binding to the second surface 170 changes the
measured amplitude response spectrum, for example by changing the
fundamental resonant frequency and also changing the harmonics or
overtones.
[0118] In a further method, the sensor head 120 is exposed a sample
that includes an analyte and is also exposed to a driving pulse or
step-function with a defined rise-time, fall-time, amplitude, duty
ratio and cycle time (repetition rate). These parameters are
selected according to the optimum response of the sensor head. The
amplitude and frequency of motion of the second surface 170 in
response to the driving pulses may be measured at the fundamental
frequency and at the harmonics or overtones. Analyte binding to the
second surface changes the measured response.
[0119] When the driving force is abruptly turned off, the second
surface oscillation amplitude decays with time. By monitoring the
decay of the second surface oscillation it is possible to measure
the dissipation, D. The speed with which the oscillation amplitude
decays is inversely proportional to the dissipation D, defined
as:
D=1/Q=(energy dissipated per cycle)/(2 pi total energy stored)
[0120] The dissipation factor may be measured every time the
driving wave generator output is stopped and the sensor oscillation
starts to decay exponentially. A soft film attached to the sensor
is deformed during the oscillation, which gives a high dissipation
while as a rigid material gives a low dissipation.
[0121] Measuring both the changes in the resonant frequency and the
dissipation factor can provide additional information about the
sample. Further, measuring changes in the resonant frequency and
the dissipation factor for multiple oscillating modes of the sensor
can provide still more information about the sample.
[0122] In a still further method, the sensor head 120 is exposed to
a sample including an analyte and the design of the sensor head is
optimized to detect changes in surface stress, for example, by
selecting the second surface dimensions and geometry as well as by
selecting the material and layer thicknesses of a single or
multilayer structure.
[0123] Analyte binding to the second surface 170 changes the
surface stress and causes a change in position, or displacement of
the second surface relative to the first surface. The displacement
of the second surface can be continuously monitored.
[0124] In a still further method, the sensor head 120 is exposed to
a sample including fluid flow with a defined flow velocity. The
speed and direction of the flow are selected according to the
optimum response of the sensor head. The motion of the second
surface 170 in response to the fluid flow is measured. Analyte
binding to the second surface changes the measured response due to
changes in effective mass, surface stress, viscosity, elasticity
and other effects.
[0125] Further, molecules and particles in the sample may impact or
collide with the sensor head 120 as they are carried by the fluid
flow. The response of the second surface 170 to the impact of the
molecules and particles can also be measured.
[0126] In a still further method the sensor head 120 is exposed to
a sample including motile components, for example sperm or
bacteria. The design of the sensor head 120 is optimized to detect
the motile components. The transient impact of one or more motile
components with the second surface may cause a detectable
deflection of the second surface 170. In addition, when the second
surface 170 is coated with an analyte-binding layer specific to the
desired motile component, the motion of the second surface 170 can
be continuously monitored when one or more of the motile components
binds and continues to move while attached to the sensor
surface.
[0127] FIG. 4 shows an embodiment of the invention including a
sensor head 120 that is removably carried on an optical fiber in
the assay apparatus. The sensor head assembly 120 includes a first
surface 160 that is capable of reflecting light and is mechanically
fixed, a second surface 170 that is capable of reflecting light and
a gap 180 separating the first and second surfaces 160, 170 that is
typically an air gap, but may be a fluid-filled gap or a vacuum.
The gap height is preferably set according to the wavelength of the
light source as described previously for maximum sensitivity to
deflections of the second surface 170. In a preferred embodiment
the second surface 170 is a thin membrane or diaphragm held in
tension by a tensioning element 215. Alternately, the spacer layer
180 attached to the second surface 170, or the first surface 160
may serve as a tensioning element. Analyte-binding molecules 190
may be immobilized on the second surface 170 such that, when the
sensor head 120 is exposed to the sample, analyte molecules 200
specifically bind to the second surface 170 with high affinity. In
this embodiment, the entire sensor head 120 may be removably
attached to an optical fiber 152 by use of a mating ferrule and
cylinder, as is well known in the art of fiber optic
connectors.
[0128] In one embodiment the second surface 170 is also removable
and can be easily replaced. This allows for a low-cost disposable
second surface 170 so that a new and fresh surface can be used for
each sample test. In another embodiment, the second surface 170 or
the entire sensor head 120 can be removed and discarded after use
to provide a fresh second surface is used for each measurement. In
another embodiment, the second surface 170 can be cleaned by
sonication, or ultrasonic cleaning, in a suitable cleaning solution
after use. The sonication process may occur in the same assay
apparatus used to make measurements simply by replacing the
measurement buffer with a cleaning solution and using a transducer
operating at appropriate amplitude and frequency to generate the
sonication cleaning energy. Alternately, the sonication process may
occur in a separate chamber specifically optimized for sensor
cleaning. If necessary, a fresh layer of analyte-binding molecules
can then be attached to the cleaned surface. In this case the
sensor head is reusable for multiple measurements.
[0129] In a further embodiment, first and second surfaces 160, 170
can be integrated into a sensing assembly, separate from the
optical fiber 152. In this case, first surface 160, second surface
170 and spacer 180 form a sensor assembly that is removably
attached to an optical fiber 152 by use of a mounting element 210.
The first surface 160 may include a substrate to form an optical
element having a single or multilayer coating that serves as the
first reflecting surface. The space between the end face of optical
fiber 152 and the first surface 160 may include an air gap. The
size of the air gap can be selected to minimize the effect of
reflections from the fiber end face. If the air gap is
significantly greater than the coherence length of the light
source, the effect of optical interference form the light reflected
by the fiber end face will be minimal. In another embodiment the
end surface of optical fiber 152 may be modified to minimize
reflected light, for example by use of a multilayer anti-reflection
coating, or by angle-polishing the end surface, by use of an
index-matching fluid or gel, or similar methods. In operation the
sensor head 120 is connected to an optical fiber 152 by use of
mounting element 210 and seated or locked in place. The sensor head
120, for example, may then be exposed to a sample including analyte
under conditions that provide for the binding of sample analyte to
the analyte-binding molecules immobilized on the second surface 170
according to the methods of the invention previously discussed.
[0130] FIG. 5A shows an alternate embodiment of the sensor
apparatus, 500, using free-space optics. In this case, a free-space
optical system 540 (which may include a variety of components such
as lenses, mirrors, beam-splitters, optical coatings, prisms,
gratings, molded optical elements and associated mounting
components) is used to direct light from the light source 510 to
the sensor head 120 and to collect the reflected light from the
sensor head and direct it to the light detector 530. This
embodiment may require tight tolerances and precise alignment of
the components of the optical system.
[0131] The light source 510, may be a LED, laser diode, solid state
laser, gas laser, and suitable wavelengths range from the DUV to
the far infrared (10 nm to 100 um). The light source 510 may emit
light primarily at a single wavelength or over a range of
wavelengths. The detector 530 may be a simple single-element
photodetector, multi-element photodetector or a detector array,
such as a charge-couple device CCD or CMOS imaging device. A single
detector element can be used to monitor the signal from the sensor
head 120. The light source 510, coupling optics 540, and light
detector 530 components are all commercially available.
[0132] In another embodiment, shown in FIG. 5B transducer 122 is
integrated into the sensor head 120 and sensor head similar to the
situation previously described in FIG. 1B. FIG. 6. shows an
embodiment of the invention including an embodiment of sensor head
120 designed for a free-space optics-based assay apparatus. The
sensor head assembly 120 includes a first surface 160 that is
capable of reflecting light and is mechanical fixed, a second
surface 170 that is capable of reflecting light and a gap 180
separating the first and second surfaces 160, 170 that is typically
an air gap but may be fluid-filled or simply a vacuum. The gap
height is preferably set according to the wavelength of the light
for the maximum sensitivity to deflections of the second surface
170 as discussed previously. In a preferred embodiment the second
surface 170 is a thin membrane or diaphragm held in tension by a
tensioning element 215. Alternately, the spacer layer 180 attached
to the second surface 170 may serve as the tensioning element.
Analyte-binding molecules 190 may be immobilized on the second
surface 170 such that, when the sensor head 120 is exposed to the
sample, analyte molecules 200 specifically bind to the second
surface 170 with high affinity. In this embodiment, the entire
sensor head 120 may be removably mounted in a carrier 230 for
alignment with the coupling optics 540 of the free-space optics
assay apparatus 500.
[0133] In one embodiment the second surface 170 is removable and
can be easily replaced. This allows for a disposable second surface
170 so that a new and fresh surface can be used for each sample
test. In another embodiment, the second surface 170, the second
surface and spacer 170, 180, the second surface, spacer and first
surface 160, 170, 180 or the entire sensor head 120 can be removed
cleaned and re-used or removed and discarded after use. In this way
a fresh second surface 170 can be used for each measurement. In
another embodiment, the second surface 170 can be cleaned by
sonication, or ultrasonic cleaning, in a suitable cleaning solution
after use. The sonication process may occur in the same assay
apparatus used to make measurements simply by replacing the
measurement buffer with a cleaning solution and using a pressure
wave transducer operating at appropriate amplitude and frequency to
generate the sonication cleaning energy. Alternately, the
sonication process may occur in a separate chamber specifically
optimized for sensor cleaning. If necessary, a fresh layer of
analyte-binding molecules can then be attached to the cleaned
surface. In this case the sensor head is reusable for multiple
measurements.
[0134] In one embodiment the first surface 160 includes a substrate
to form an optical element having a single or multilayer coating
that serves as the first reflecting surface. The space between the
coupling optics and the first surface 160 may include an air gap.
The size of the air gap can be selected to minimize the effect of
undesired reflections from optical elements of the sensing
apparatus. If the air gap is significantly greater than the
coherence length of the light source, the effect of optical
interference form the light reflected by the fiber end face will be
minimal. In another embodiment the optical elements of the sensing
apparatus may be modified to minimize reflected light, for example
by use of a multilayer anti-reflection coating, or by
angle-polishing the end surface, by use of an index-matching fluid
or gel, or similar methods.
[0135] In operation the sensor head 120 is aligned with the
free-space optical system 500 by use of mounting element 230 and
seated or locked in place. The sensor head 120 can then exposed to
a sample of analyte under conditions that provide for the binding
of sample analyte to the analyte-binding molecules immobilized on
the second surface 170 according to the methods of the invention
previously discussed.
[0136] FIG. 7 shows an alternate embodiment of the sensor apparatus
700 using a laser diode sensor. This embodiment includes a laser
diode 710 having two light-emitting end-faces 720 and 730. The
laser diode 710 is carried by a mount 740 and positioned proximate
the sensor 120 with the laser diode light emitting face 730
oriented to direct light to the sensor 120 and receive light from
the sensor 120. A light detector 750 is mounted close adjacent the
other face of the laser diode 720 for receiving laser light. The
light detector 750 generates an electrical signal directly related
to the power of the laser light and provides a measure of the
displacement of the second surface 170 relative to the first
surface 160 of the sensor head 120 by use of optical interference
as described previously. The gap between the first surface 730 and
second surface 170 is typically in the range 1 um (10-6 meters) to
1 cm (10-2 meters). Although the light detector 750 is shown
separate from the laser diode 710, it is also possible to produce
an integrated device combining both light detector 750 and laser
diode 710 into a single unit.
[0137] A solid state laser comprises a cavity in which light is
generated and made to reflect from end to end. Gain is added to the
cavity such that the energy of the light continues to increase
until the device "lases", i.e. the light energy is sufficient to
pass through the reflective surfaces 720, 730 to produce a beam of
laser light directed to the second surface of the sensor 170. The
light reflected by second surface 170 back into the laser diode 710
adds to the light reflected internally from the front face 730 in a
manner which depends on the relative phase. Light reflected by the
second surface 170 can add either constructively or destructively
with the light in the laser diode cavity and this will vary with
the distance between second surface 170 and the laser diode front
face 730. Some of the laser diode light output is transmitted by
the laser diode surface 720 to the light detector 750 to provide a
measure of the laser diode light power output.
[0138] The effect of optical interference between light reflected
by the two surfaces, 170 and 730 is to produce a variation of the
laser diode light power output with gap similar to the variation
shown in FIG. 3. When the light reflected by second surface 170 is
in-phase with the light reflected by the laser diode front face
730, the laser diode output from face 720 will be a maximum and
when the light from the second surface 170 is out-of-phase, the
lasers output will be a minimum. If the second surface is moved
one-quarter of a wavelength of the laser light, either toward or
away from the laser diode front face 730 the output will vary from
a maximum to a minimum as depicted in the figure. Using this
optical interference effect, displacements of the second surface
170 can be detected by monitoring the changes in the laser output
power as sensed by the light detector 750. If the laser wavelength
is .about.1 micron (10-6 meters) this distance is about 250
nanometers, as previously described. To maximize sensitivity, the
modulation of laser diode power due to second surface displacement
should be as large as possible. This can be achieved by optimizing
the position, alignment and reflectivity of all optical
surfaces.
[0139] FIG. 8 illustrates a sensor assembly in an embodiment of the
invention designed for detecting one or more of a plurality of
analytes in a sample. In the case of a sensor apparatus using
optical fibers, individual optical fibers may be mounted in a
carrier, for example a multi-fiber ferrule, to provide for precise
alignment of both position and angle for each fiber in the array.
The array may be linear (1-dimensional) or aerial (2-dimensional).
Alternately the individual fibers may be fused to form a fiber
bundle. The sensor assembly 800 is composed of the same elements
described previously for the sensor head in FIG.4, but in an array
format. In one embodiment, an array of first reflecting surfaces
160 may be formed on the fiber end surfaces. Further, an array of
second surfaces, 170 each of which is movable in response to an
applied force or pressure, is positioned near the first reflecting
surfaces and spaced by a gap. A spacer layer 180 having a thickness
selected for optimum sensitivity is sandwiched between the first
and second surfaces 160, 170 to define a gap. In a preferred
embodiment the second surface 170 is a thin membrane or diaphragm
held in tension by a tensioning element 215 in a mount 220.
Alternately, the spacer layer 180 attached to the second surface
170, or the first surface 160 may serve as a tensioning element.
Each of the second surfaces 170 may have an immobilized layer of
analyte-binding molecules 190 specific for one or more of the
analytes in the sample.
[0140] In one embodiment, the array of second surfaces 170 is
removably mounted and aligned to the fibers in the fiber array by
use of a mechanical mount that mates to the multi-fiber ferrule or
fiber bundle. In this way, each of the optical fibers is aligned
with an element in the sensor array 800 and each fiber is directing
light to and receiving light from one of the elements in the sensor
array.
[0141] As previously discussed with reference to FIG. 4, a second
optical element having a single or multilayer coating can also
serve as the first reflecting surface 160. In this case, the space
between the end face of optical fiber 152 and the first reflecting
surface 160 also includes a gap. The size of the gap 180 can be
selected to minimize the effect of reflections from the optical
fiber end-face. If the gap 180 is significantly greater than the
coherence length of the light source, the effect of optical
interference form the light reflected by the optical fiber end face
will be minimal. In another embodiment the optical fiber end
surfaces may be modified to minimize reflected light, for example
by use of a multilayer anti-reflection coating, or by
angle-polishing the end surfaces, by use of an index-matching fluid
or gel, or similar methods.
[0142] In addition, the optical coupler in the apparatus which
serves to couple the fibers in the array to both the light source
and light detector directs reflected light from each element in the
sensor array to a corresponding area on a detector array, for
example a two-dimensional CCD, a CMOS detector array or a PIN
photodiode array. In this way, the signal from each element in the
sensor array can be monitored by a corresponding element in the
light detector array.
[0143] In an alternate embodiment, the sensor apparatus 800 may
include free-space optics. In this case, the sensor apparatus
includes a free-space optical system similar to the one described
previously in FIG. 5. The free-space optics sensor apparatus is
used to direct light from one or more light sources to the sensor
elements and to collect the reflected light from the sensor
elements and direct it to one or more light detectors. This
embodiment may require tight tolerances and precise alignment of
the components of the optical system.
[0144] The light source or sources may be an LED, laser diode,
solid state laser, gas laser, and suitable wavelengths range from
the DUV to the far infrared (10 nm to 100 um). The light source or
sources may emit light primarily at a single wavelength or over a
range of wavelengths. The light detectors may be a simple
single-element photodetector, multi-element photodetector or a
detector array, such as a charge-couple device CCD or CMOS imaging
device. The light source, coupling optics, and light detector
components are all commercially available.
[0145] The embodiment of the invention using a laser diode sensor
previously described in FIG. 7 can also be extended for the
detection of one or more of a plurality of analytes in a sample by
use of multiple laser diodes, each having a separate mount, or
sharing a common mount. Further, it is also possible to use a laser
diode bar including multiple laser diode elements.
[0146] In one application the sensor array 800 forms a "gene chip"
for detecting a plurality of different gene sequences. Each sensing
element in the array has an immobilized DNA sequence designed to
specifically hybridize with a complementary DNA sequence in the
sample. More generally, applications of the apparatus of this
invention include: [0147] 1. Screening hybridoma expression lines.
[0148] 2. Characterizing antibody affinity to an antigen. [0149] 3.
Characterizing protein binding partners, including DNA, RNA,
proteins, carbohydrates, organic molecules. [0150] 4.
Characterizing binding partners including DNA, RNA, proteins,
carbohydrates, organic molecules. [0151] 5. Characterizing binding
of the components in a protein that participates in a multi-protein
complex attached to the sensor. [0152] 6. Characterizing binding
partners for a protein binding molecule attached to the sensor.
Constructing a calibration curve for analyte using a set of analyte
standards. Using the calibration curve to determine the analyte
concentration in unknown solutions. [0153] 7. Identifying specific
binding partners for single-stranded DNA or RNA attached to the
sensor. [0154] 8. Single nucleotide polymorphism analysis. [0155]
9. Gas sensing (for example, an artificial "nose") [0156] 10.
Measuring, monitoring, detecting and characterizing the deposition
of thin films, in liquid, in air or in vacuum. [0157] 11.
Measuring, monitoring, detecting and characterizing adsorption,
moisture, particulate, contamination, bubble formation, surface
oxidation, and corrosion in liquid, in a gaseous environment or in
vacuum. [0158] 12. Detecting of virus capsids, bacteria, mammalian
cells, biomembranes, biomaterials, self-assembled monolayers,
molecularly imprinted polymers, langmuir-blodgett films, materials
characterization and monitoring.
* * * * *