U.S. patent application number 10/266170 was filed with the patent office on 2003-06-19 for refractometer and method for qualitative and quantitative measurements.
Invention is credited to Atkinson, Robert C., Byrne, Michael J., Ryan, Thomas E..
Application Number | 20030112427 10/266170 |
Document ID | / |
Family ID | 26805867 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030112427 |
Kind Code |
A1 |
Ryan, Thomas E. ; et
al. |
June 19, 2003 |
Refractometer and method for qualitative and quantitative
measurements
Abstract
A sensor apparatus and associated method for sensing and
monitoring specific binding of analyte to an immobilized binding
layer are disclosed. The apparatus preferably comprises an
automatic critical angle refractometer having a linear scanned
array and an optical system for illuminating a portion of the
array, which illumination depends upon the refractive index of the
binding layer deposited on an optically transparent element. The
apparatus further includes a flow cell for bringing the analyte in
contact with the binding layer. The apparatus also includes a
computer for receiving and processing refractive index data from
the critical angle refractometer during the reaction between the
analyte and the layer, which computer may be peripherally connected
to the refractometer or enclosed within the refractometer housing.
A preferred sensing method of the present invention generally
comprises providing a critical angle refractometer generating light
impinging upon the immobilized binding layer, contacting the
binding layer with a contacting phase, measuring the critical angle
of total reflection, which measurements are indicative of the
presence or absence of interactions between the analyte and the
binding layer.
Inventors: |
Ryan, Thomas E.; (Batavia,
NY) ; Byrne, Michael J.; (East Aurora, NY) ;
Atkinson, Robert C.; (Buffalo, NY) |
Correspondence
Address: |
BROWN, RUDNICK, BERLACK & ISRAELS, LLP.
BOX IP, 18TH FLOOR
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
26805867 |
Appl. No.: |
10/266170 |
Filed: |
October 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10266170 |
Oct 7, 2002 |
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09439876 |
Nov 12, 1999 |
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6462809 |
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60108414 |
Nov 13, 1998 |
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60142207 |
Jul 2, 1999 |
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Current U.S.
Class: |
356/128 ;
356/136 |
Current CPC
Class: |
G01N 21/05 20130101;
B82Y 30/00 20130101; Y02A 90/10 20180101; G01N 33/551 20130101;
G01N 33/54373 20130101; G01N 21/43 20130101; G01N 33/544
20130101 |
Class at
Publication: |
356/128 ;
356/136 |
International
Class: |
G01N 021/41 |
Claims
What is claimed is:
1. A method of using critical angle refractometry for sensing
presence or absence of an analyte at a binding layer, the method
comprising: providing a first optically transparent element and a
second optically transparent element, the first optically
transparent element having a higher refractive index than that of
the second optically transparent element, the second element having
the binding layer; providing a contacting phase; allowing the
contacting phase to contact the binding layer of the second
optically transparent element; passing light through the first and
the second optically transparent elements to cause the light to
impinge upon an interface between the second optically transparent
element and the binding layer; and detecting a location of a
boundary between a light area and a dark area on a sensing element,
the location of the boundary being indicative of the presence or
absence of the ligands at the binding layer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a Continuation of application Ser. No.
09/439,876 filed on Nov. 12, 1999 which claims the benefit from
U.S. provisional patent application Serial No. 60/108,414, filed
Nov. 13, 1998, and U.S. provisional patent application Serial No.
60/142,207, filed Jul. 2, 1999, which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
refractive index based sensing devices. More particularly, the
invention relates to a critical angle refractometer and method for
sensing and monitoring interactions between an analyte and a
binding layer.
BACKGROUND OF THE INVENTION
[0003] Analysis of qualitative and quantitative aspects of
interactions between analytes and various types of binding layers
is important to a wide range of scientific and industrial
applications. Consequently, sensors which monitor specific binding
of a sample analyte to a particular type of ligands immobilized on
the sensing surface have been developed. The term "ligands" here
means a type of molecules exhibiting specific binding affinity to
another type of molecules. The terms "immobilized binding layer",
"binding layer", or "sensing layer" here mean a layer formed by
ligands immobilized on a sensing surface. The term "sensing
surface" means an interface between two media, one of which is the
binding layer. The term "contacting phase" here means a fluid
phase, which is brought in contact with the binding layer. The term
"analyte", or "sample analyte", here means the ligands contained in
the contacting phase. An analyte in a contacting phase may or may
not possess binding affinity to a particular binding layer.
[0004] For example, sensors based on the surface plasmon resonance
(SPR) phenomenon are known to detect and measure changes in the
refractive index of a sample analyte contacting a sensing layer.
SPR sensors are often used in such applications as investigation of
surface and interface effects, spectroscopy, differential
reflectivity, immunoasays. SPR sensors are based on the following
principle: when a thin metal layer is illuminated by an incident
beam of light, under certain circumstances the energy of the light
beam can excite free electrons on the illuminated surface of the
metallic film. In particular, the beam will resonate with the
surface electrons, which resonance will lead to the creation of an
electrical field extending within the range of about 200
nanometers. The resonance occurs at a certain angle of incidence of
the incoming light beam and depends on the refractive index of a
substance located within the range affected by the generated
electrical field. Binding or dissociation of the analyte and an
immobilized binding layer at the sensor surface changes the local
refractive index at the surface and produces a shift in the
resonant angle of incidence, which has been shown to be
proportional to the concentration of ligands bonded to an
immobilized binding layer up to a predetermined limiting
concentration. Thus, by electro-optically monitoring changes of the
refractive index at the sensing surface using SPR, qualitative
sensing of ligands and quantitative characterization of various
binding kinetics and equilibria are possible.
[0005] An example of an SPR biosensor is schematically illustrated
in FIG. 1. SPR biosensor 2 includes a prism 4 having a test surface
thereof coated by a thin metallic film 6. A first type of ligands 8
is immobilized on metallic film 6, and an analyte 10 is introduced
into the contacting phase above the test surface. A light source 12
of predetermined wavelength directs an incident beam 14 to metallic
film 6, and a photosensitive detector 16 is arranged to monitor the
intensity of reflected beam 14'. At a certain angle of incidence
.alpha. of beam 14, resonant excitation of electrons (surface
plasmons) in metallic film 6 results in absorption of incident beam
14 and, consequently, in an energy loss in the reflected beam 14',
which is observed experimentally as a sharp minimum in the
intensity of light received by detector 16, as illustrated in FIG.
2.
[0006] While SPR sensors exhibit a high degree of sensitivity to
changes in refractive indices, which makes them a useful research
tool, immobilizing a binding layer on a metallic layer is both
difficult and limiting. It is difficult, because the immobilization
technique must attach the ligands in a native conformation and in a
uniformly reactive and accessible orientation, to a metallic
surface that does not allow for a significant amount of
non-specific binding. A number of various immobilization techniques
have been described in the art, with the choice of a technique
being dependent upon particular ligands involved. Because of these
and other difficulties associated with manufacturing SPR sensors,
such sensors are expensive. Therefore, it would be desirable to
come up with a less expensive device capable of measuring changes
of the refractive indices caused by interactions between various
ligands.
[0007] An example of a suitable device for sensitive and
quantitative measurements associated with changes in refractive
indices is a critical angle refractometer. The operation of a
critical angle refractometer is based on the following principle.
When light is incident on a surface separating two media, the light
is refracted at the interface between the two media in accordance
with Snell's law:
n Sin I=n' Sin I'
[0008] where n and n' are the refractive indices of the two media,
and I and I' are the angles of incidence and refraction,
respectively. Light can always pass from a lower refractive index
medium to a higher refractive index medium, because in that case
angle I' is smaller than angle I. However, when a beam of light
passes from an optically denser medium (having a higher index of
refraction n) to an optically rarer medium (having a lower index of
refraction n'), the angle of refraction I' is always greater than
the angle of incidence I. As the angle of incidence I increases,
the angle of refraction I' increases at a faster rate. When Sin
I=n'/n, then Sin I'=1.0 and the angle of refraction I'=90 degrees.
Such an angle of incidence is called the critical angle. When the
critical angle condition is met, no light propagates into the
optically rarer medium. When the angle of incidence is greater than
the critical angle, the light is reflected back into the optically
denser medium--a phenomenon called total internal reflection
(T.I.R.). If the separating boundary of the two media is smooth and
clean, 100 percent of the incident light is reflected back. The
critical angle phenomenon is used for measurements of refractive
indices of various fluid or solid materials.
[0009] FIG. 3a depicts a critical angle refractometer shown and
identified broadly by the reference numeral 22. Refractometer 22 is
shown as including a housing 32 having an inclined top surface
portion 34 and a horizontal top surface portion 36 adjacent
thereto, an LCD display 38 and a keypad input 40 at inclined top
surface portion 34. A test assembly 24 is situated on horizontal
top surface portion 36. Refractometer 22 is similar to the Leica
AR600 automatic refractometer available from Leica Microsystems
Inc. The Leica AR600 automatic refractometer is manufactured
generally in accordance with the disclosure of commonly-owned U.S.
Pat. No. 4,640,616 issued Feb. 3, 1987 and entitled AUTOMATIC
REFRACTOMETER. The entire disclosure of U.S. Pat. No. 4,640,616 is
incorporated herein by reference as if reprinted in its
entirety.
[0010] The schematic of FIG. 4 illustrates the opto-electronic
measurement system of refractometer 22, which is based on the
principles of critical angle refractometry described above. The
system comprises a photosensitive linear scanned array (LSA) 44 for
providing an output signal as a function of the amount and location
of light incident thereon. Linear scanned array 44 includes a
plurality of closely adjacent and aligned photoelectric cells 46.
The measurement system comprises an optical system for directing
light onto linear scanned array 44, wherein the amount and location
of light illuminating the LSA depends on the index of refraction of
a test sample 51. As shown in FIG. 4, the optical system includes a
light source 48 and a prism 50 for receiving light along an optical
path 57 from source 48. Prism 50 includes a top surface 54 for
receiving test sample 51, a bottom surface 56 parallel to top
surface 54 through which light enters and exits the prism, and a
pair of internally reflective side surfaces 58 and 60, which define
acute included angles with bottom surface 56. A temperature sensor
52 is provided at top surface 54 to read sample temperature for
temperature compensation purposes.
[0011] Light originating from source 48 travels sequentially
through a diffuser 62, a polarizer 64, and a collimating lens 66.
The parallel light leaving collimating lens 66 enters an
interference filter 68 which transmits essentially monochromatic
light at a wavelength of 589 nm. A converging lens 70 is arranged
to receive light transmitted by filter 68 and concentrate the light
in the direction of a reflecting mirror 72, which is orientated to
reflect the light through the bottom surface 56 of prism 50. The
light is totally internally reflected by side surface 58 to impinge
upon top surface 54. A first portion of light (not shown) incident
on top surface 54 at the angles less than the critical angle is
refracted into sample 51. A second portion of light 55 incident on
surface top 54 at the angles larger than the critical angle is
totally internally reflected from top surface 54. Second portion of
light 55 is then internally reflected by side surface 60 and exits
prism 50 through bottom surface 56. After passing through a lens
73, portion 55 is redirected by a reflecting mirror 74 in the
direction of linear scanned array 44. Therefore, light distribution
at LSA 44 consists of an illuminated region 47, formed by second
portion of light 55, and a non-illuminated region 47a. The boundary
between the two regions 47 and 47a is referred to as the shadow
line, and its position on linear scanned array 44 is dependent upon
the refractive index of test sample 51.
[0012] In the Leica AR600 automatic refractometer, the LSA contains
almost 2600 individual charge-coupled device (CCD) elements, each
of which is a 11 .mu.m.sup.2square. Each CCD, pixel, is capable of
converting the intensity of light hitting upon it into an
electrical voltage, which is subsequently converted to a digital
number between 0 and 255 by supporting circuitry. Each CCD produces
a numeric intensity value as an output reading. A typical graph,
illustrating illumination intensity from a bare prism (a reference
reading of air) as a function of a cell number, is shown in FIG.
5a. The reference reading of air in FIG. 5a is taken by pressing an
INITIATE key of keypad input 40 to provide a reference curve 100,
corresponding to the illumination distribution at linear scanned
array 44 without a sample on top surface 54 of prism 50. When a
sample is placed on the prism, the first portion of the light is
transmitted through the sample, and second portion of light 55 is
reflected toward the LSA, illuminating a part of it, thus, forming
a shadow line on the LSA, as described above with regard to FIG. 4.
Determination of the shadow line location expressed as the
crossover cell number is carried out by a software routine stored
in the programmable memory of refractometer 22. During a reading,
reference curve 100 is scaled by 94%, as indicated by the dashed
curve just below reference curve 100 in FIG. 5b, forming a scaled
reference curve 120. The scaling parameter does not have to be 94%,
it can vary (80%, 85% for example) to achieve the best precision
between consecutive readings. The crossover cell number is then
found by a routine, which identifies the cell or cell fraction at
which a sample curve 110 intersects with scaled reference curve
120. The crossover cell number is then converted to a refractive
index value, based on a calibration reading of a substance of a
known refractive index.
[0013] Despite the fact that the critical angle reflection
phenomenon has been known in the past, there has been no successful
effort to bring critical angle refractometers into the analytical
art as sensors, capable of detecting and monitoring binding between
an analyte and a binding layer having specific affinity to the
analyte. Since critical angle refractometers, such as, for example,
the above-described Leica AR600 automatic refractometer are
inexpensive, compared to commercially available SPR sensors, it
would be desirable to use a critical angle refractometer to sense
and monitor binding phenomena. Therefore, the need exists to
provide a method and device utilizing critical angle refractometry
to sense and monitor the presence and the amount of a particular
analyte by measuring changes in the refractive index occurring due
to specific binding of the analyte to an immobilized binding layer
on a sensing surface.
SUMMARY OF THE INVENTION
[0014] Therefore, it is an object of the present invention to
provide a sensing device and method utilizing critical angle
refractometry to sense and monitor binding interactions between a
sample analyte and a binding layer.
[0015] It is another object of the present invention to provide a
sensor device, which does not measure changes in a refractive index
by using the surface plasmon resonance phenomenon, and thus avoids
the need for experimentally rigorous procedure of immobilization of
a binding layer on a thin metallic layer. A related object of the
invention is to avoid problems associated with oxidation of a
metallic layer and the necessity to provide an intermediate layer
between the metallic layer and a glass surface in traditional SPR
sensors.
[0016] It is a further object of the present invention to provide a
critical angle based sensor device, which is affordable to
manufacture and simple to operate.
[0017] It is yet another object of the present invention to provide
a critical angle refractometric method and apparatus for measuring
changes in the refractive index at a sensing layer by passing light
through an optically transparent arrangement to cause the light to
be totally internally reflected at the sensing layer.
[0018] It is also another object of the present invention to
provide a method and device utilizing critical angle refractometry
to sense presence or absence of a sample analyte in a contacting
phase by measuring the critical angle of total reflection of light
at a sensing layer.
[0019] It is yet another object of the present invention to provide
a critical angle refractometer and method for measuring the rate of
a binding reaction between a binding layer and a sample
analyte.
[0020] In view of these and other objects, an apparatus and method
for sensing presence and the amount of an analyte in a contacting
phase are provided by using a critical angle refractometer to sense
changes in a refractive index of a sensing layer occurring as the
interaction between the sample analyte and an immobilized binding
layer progresses over time. The apparatus, according to one of the
embodiments of the present invention, comprises an automatic
critical angle refractometer for obtaining refractive index data
with respect to a sample analyte in operative association with an
opto-electronic measurement system of the refractometer, and a
computer connected for data communication with the refractometer
for processing the data and reporting changes in the refractive
index as a function of time.
[0021] The refractometer measurement system includes a linear
scanned array of photosensitive cells, and an optical system for
directing light onto the LSA. The light impinging upon the LSA
forms a shadow line, dividing the LSA into an illuminated portion
and a dark non-illuminated portion. The location of the shadow line
is dependent on the refractive index of a binding layer immobilized
on the sensing surface. Depending on whether the sample analyte has
bonded with the binding layer, the position of the shadow line will
change. Therefore, correlation of the shadow line location to a
value, which is a function of the refractive index, such as a
concentration of the sample analyte in the contacting phase, can be
established. The correlation is carried out by software routines
stored in the programmable memory of the refractometer.
[0022] In accordance with the present invention, a method of using
critical angle refractometry for sensing and monitoring
interactions between the analyte and the binding layer is provided.
An optical system directs light through one or more optically
transparent elements to impinge upon the interface between the
binding layer and one of the optically transparent elements. The
absence or presence of binding between the analyte and the binding
layer changes the refractive index of the binding layer. The
refractive index of the binding layer, in turn, affects the
critical angle of total reflection. The light reflected from the
interface at a particular angle impinges on the LSA, creating a
shadow line, the location of which can be related to the amount of
the analyte bonded to the immobilized binding layer. The same
principle enables the method and apparatus of the present invention
to monitor and measure the rate of changes in the refractive index,
which rate is proportional to the concentration of the analyte in a
contacting phase and the strength of affinity between the analyte
and the binding layer.
[0023] The present invention also provides an apparatus and method
for sensing the presence or absence of a particular analyte having
specific affinity to the binding layer by measuring changes of the
refractive index at the binding layer. Such sensing can be
implemented in laboratory tests and home test kits. The method
comprises directing a collimated light beam at a particular
incident angle through one or more optically transparent elements
to impinge upon the interface between the binding layer and one of
the optically transparent elements. Depending on whether a
particular analyte with specific affinity to the binding layer is
present or absent in the contacting phase, the incident angle of
light will or will not satisfy the condition for total internal
reflection. If the condition for total internal reflection is
satisfied, the reflected light will impinge on the LSA or any other
sensor capable of detecting light, disposed along the optical path
of the reflected light. Therefore, depending on whether the sensor
is illuminated by totally internally reflected light, the presence
or absence of the analyte can be determined. It is also
contemplated that the LSA can be disposed to sense transmitted
light, which will illuminate the LSA depending on whether the
T.I.R. condition is satisfied. An apparatus for practicing the
above-described method comprises a collimated beam of light
directed at the interface at a particular angle of incidence. In
order to sense the critical angle of total reflection, a single
light source capable of moving and changing the angle of incidence
is provided. In an alternative embodiment of the apparatus, a
plurality of light sources directing light beams at the interface
at different angles, are utilized to sense the presence or absence
of the analyte. Depending on whether the binding between the
analyte and the immobilized binding layer has occurred, the light
from one of the light sources becomes totally internally reflected
at the interface, therefore, illuminating the light sensor and
indicating the presence or absence of the analyte.
[0024] In one of the embodiments of the invention, a specialized
test assembly allows for operative association between the sample
analyte in a contacting phase and the immobilized binding layer. In
the preferred embodiment, the apparatus includes a thin, optically
transparent element having a selected type of ligands immobilized
on an upper surface thereof, forming a binding layer. A flow cell
is arranged closely above the transparent element for providing a
buffer flow of the contacting phase containing the sample analyte
intended for specific binding interaction with the immobilized
binding layer. An 0-ring or a gasket arranged on the upper surface
of the disc is sized to provide a peripheral fluid-tight seal
between the binding layer on the sensing surface of the element and
the flow cell. A high refractive index coupling liquid is provided
between a lower surface of the optically transparent element and
the top surface of the refractometer prism. The transparent
elements, such as discs, are preferably formed of glass,
polystyrene, polycarbonate, or other optically transparent
materials with a suitable index of refraction. A particular
immobilization technique usually depends in part on the material
used to form the disc. By way of example, an antibody, such as an
anti-strepavidin antibody, may be immobilized on the upper surface
of the optically transparent disc, and its antigen strepavidin
introduced in a buffer flow for analysis of binding interactions.
By way of further example, with respect to DNA binding protein/DNA
ligand interactions, the OccR protein may be immobilized on the
upper surface of the optically transparent disc, and its
oligonucleotide target introduced in a contacting phase for
analysis of binding interactions.
[0025] To summarize, the present invention provides a method of
using critical angle refractometry for sensing presence or absence
of an analyte at a binding layer, the method comprising providing a
first optically transparent element and a second optically
transparent element, the first optically transparent element having
a higher refractive index than that of the second optically
transparent element, the second element having the binding layer,
providing a contacting phase, allowing the contacting phase to
contact the binding layer of the second optically transparent
element, passing light through the first and the second optically
transparent elements to cause the light to impinge upon an
interface between the second optically transparent element and the
binding layer, and detecting a location of a boundary between a
light area and a dark area on a sensing element, the location of
the boundary being indicative of the presence or absence of the
ligands at the binding layer. The method further provides a contact
layer coupling the first optically transparent element to the
second optically transparent element. The contacting phase can be
liquid, the second optically transparent element is selected from
the group consisting of glass and plastic. The binding layer is
selected from the group consisting of carboxymethylated dextran,
aldehyde activated dextran, hydrazide activated dextran, silanated
surfaces, silanized surfaces, silane, aviden, streptaviden,
neutraviden, biotinyl, bifunctional spacer arms, self assembled
monolayers, lipids and unchanged or uncoated surface of the second
optically transparent element. The contacting phase containing the
analyte comprises selecting the analyte from the group consisting
of antigens, proteins, glycoproteins, vitamins, microbes, pieces of
microbes including bacteria and bacterial fragments, viruses,
pieces of viral material, lipids, carbohydrates, toxins, DNA, RNA,
DNA and RNA analogs, pathogenic organic molecules, anti-bacterial
and anti-viral organic molecules and their analogs, therapeutic
agents and drugs.
[0026] Another embodiment of the invention is a method of using
critical angle refractometry for sensing presence or absence of an
analyte at a binding layer of a first optically transparent
material, the method comprising providing the first optically
transparent material of a higher optical density than that of the
binding layer contacting the binding layer with a contacting phase
passing light along an optical path through the first optically
transparent material to cause the light to impinge upon an
interface between the binding layer and the first optically
transparent material sensing a boundary between a light area and a
dark area on a sensing element disposed along the optical path, and
utilizing the location of the boundary to determine the presence or
absence of the analyte at the binding layer. The optically
transparent material is selected from the group consisting of glass
and plastic.
[0027] Yet another embodiment of the invention is a method for
sensing presence or absence of an analyte at a binding layer, the
method comprising providing an interface between the binding layer
and an optically transparent element, the interface being located
along an optical path, the binding layer and the optically
transparent element having different optical densities sufficient
to totally internally reflect light impinging on the interface,
contacting the binding layer with a contacting phase, illuminating
the interface with the light propagating along the optical path, so
that a portion of the light totally internally reflected from the
interface propagates between the interface and a sensing element
disposed along the optical path and illuminates the sensing element
to form a light area thereon, and detecting a location of a
boundary between the light area and a dark area on the sensing
element, the location of the boundary being indicative of the
presence or absence of the analyte at the binding layer. The
portion of the light propagating between the interface and the
sensing element comprises light reflected from the interface or
transmitted through the interface.
[0028] Another embodiment of the invention is a method of sensing
presence or absence of an analyte at a binding layer comprising
providing a light beam generated by a light source, providing an
interface between the binding layer and an optically transparent
element, the binding layer and the optically transparent element
having optical densities sufficient to cause the light beam
impinging upon the interface to be totally internally reflected,
contacting the binding layer with a contacting phase, illuminating
the interface by the light beam impinging upon the interface at a
predetermined angle of incidence, providing a sensor located at a
position in which the sensor can sense the light totally internally
reflected at the interface, and sensing the presence or absence of
light by the sensor, the presence or absence of light being
indicative of the presence or absence of the analyte at the binding
layer. The method further comprises altering the predetermined
angle of incidence by moving or rotating the light source or by
moving or rotating the optically transparent element. The method
also comprises providing a plurality of light sources so that
altering the angle of incidence is accomplished by illuminating the
interface by a light beam from a different light source. The
described sensor can comprise a plurality of sensing elements.
[0029] And yet another embodiment of the invention is a system for
detecting presence or absence of an analyte in a contacting phase,
the system comprising an optically transparent element having a
binding layer deposited thereon, the binding layer having affinity
to the analyte a critical angle refractometer defining an optical
path of a collimated light beam impinging upon an interface between
the binding layer and the optically transparent element, the
contacting phase contacting the binding layer, and a sensor
disposed along the optical path to detect changes in an optical
density of the binding layer by sensing light travelling along the
optical path. The system further comprises a test assembly serving
to bring the contacting phase in contact with the binding layer,
wherein the optically transparent element is a disposable slide and
wherein the contacting phase is a biological fluid. The system
further comprises a plurality of light sources, wherein each light
source is capable of directing a light beam toward the interface at
a predetermined angle of incidence and wherein the sensor comprises
a plurality of sensing elements.
[0030] The present invention also encompasses a method of
monitoring specific binding during a particular reaction involving
an analyte, the method comprising immobilizing a binding layer on
an optically transparent element; bringing the transparent element
into operative association with an opto-electronic measurement
system of an automatic critical angle refractometer, introducing a
contacting phase containing the analyte to contact the binding
layer, using the critical angle refractometer to generate
measurement data, including data that are a function of the
refractive index of the binding layer, at regular intervals over
time, and processing the measurement data to permit analysis of the
progress of specific binding of the analyte to the binding
layer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The nature and mode of operation of the present invention
will now be more fully described in the following detailed
description of various embodiments taken with the accompanying
drawing figures, in which:
[0032] FIG. 1 is a schematic representation of an SPR sensor;
[0033] FIG. 2 is a graph of intensity of an SPR sensor as a
function of angle of incidence;
[0034] FIG. 3a is a view of a critical angle refractometer;
[0035] FIG. 3b is a perspective view of a sensor apparatus
according to the present invention, including an automatic critical
angle refractometer;
[0036] FIG. 4 is a schematic representation of an opto-electronic
measurement system of the automatic refractometer shown in FIG.
3a;
[0037] FIG. 5a is a graph illustrating a reference curve of a
refractometer initiated in air;
[0038] FIG. 5b is a typical graph of illumination intensity as a
function of array cell number;
[0039] FIG. 6 is a graph illustrating a calibration method of the
present invention;
[0040] FIG. 7 is a schematic exploded view of the preferred test
assembly of the present invention;
[0041] FIG. 7a is a perspective view of a base and a plate of an
embodiment of the present invention;
[0042] FIG. 7b is a perspective view of a base and a plate with a
slide;
[0043] FIG. 7c is a perspective view of a flow cell cap;
[0044] FIG. 7d is a perspective view of an assembled flow
cell/slide embodiment;
[0045] FIG. 7e is an exploded view of the embodiment illustrated in
FIGS. 7a-7d;
[0046] FIG. 8 is a graph illustrating the effect of sucrose
addition on the refractive index;
[0047] FIG. 9 is a graph illustrating the effect of Bovine Serum
Albumin addition on the refractive index;
[0048] FIG. 10a is a graph illustrating the change in refractive
index over time, sensed by measuring the critical angle, the change
resulting from binding between Rabbit Anti-Sheep Antibody
immobilized on a Xenobind glass slide and Sheep IgG Antigen in the
contacting solution;
[0049] FIG. 10b is a number of graphs containing fitted data
corresponding to FIG. 10a;
[0050] FIG. 11a is a graph illustrating the change in refractive
index over time, sensed by measuring the critical angle, the change
resulting from binding between Goat Anti-Mouse Antibody immobilized
on a Xenobind glass slide and Mouse IgG Antigen in the contacting
solution;
[0051] FIG. 11b is a number of graphs containing fitted data
corresponding to FIG. 11a;
[0052] FIG. 12 is a graph illustrating the effect of binding
between biotinylated glass and neutraviden conjugated Goat
Anti-Mouse Antibody on the refractive index;
[0053] FIG. 13a is a graph illustrating the effect of non-binding
and binding ligands on the refractive index;
[0054] FIG. 13b is a number of graphs containing fitted data
corresponding to FIG. 13a;
[0055] FIG. 14 is a cross-sectional view of an arrangement used for
restricting chemical activation to only one side of a transparent
disc used to support an immobilized reactant;
[0056] FIG. 15a is a schematic illustration of the optical system
of one embodiment of the present invention;
[0057] FIG. 15b is a schematic illustration of the optical system
of another embodiment of the present invention;
[0058] FIG. 15c is a schematic illustration of yet another
embodiment of the present Invention;
[0059] FIG. 16a is a graph illustrating changes in the refractive
index over time, sensed by measuring the critical angle, the change
resulting from binding between a biotinylated glass slide and
Neutraviden conjugated Goat Anti-Mouse IgG Antigen in the
contacting solution;
[0060] FIG. 16b is a graph illustrating changes of light intensity
in the trial corresponding for FIG. 16a;
[0061] FIG. 16c is a graph illustrating changes in the position of
the shadow line in the trial corresponding to FIG. 16a;
[0062] FIG. 16d is a graph illustrating the position of the shadow
line at the beginning of the trial corresponding to FIG. 16a;
[0063] FIG. 17a is a perspective top view of a slide of the present
invention;
[0064] FIG. 17b is a perspective bottom view of the slide depicted
in FIG. 17a;
[0065] FIG. 17c is a bottom view of the slide;
[0066] FIG. 17d is a top view of the slide;
[0067] FIG. 17e is a cross sectional side view of the slide
depicted in FIGS. 17a-17e.
DETAILED DESCRIPTION OF THE INVENTION
[0068] In the following detailed description of the invention,
reference is made to the accompanying drawings, which form a part
hereof, and in which specific preferred embodiments for practicing
the invention are shown by way of illustration. These embodiments
are described in sufficient detail to enable those skilled in the
art to practice the invention, and it is to be understood that
other embodiments may be utilized and that logical, mechanical,
chemical and electrical changes may be made without departing from
the spirit and scope of the present invention. The following
detailed description is, therefore, not to be taken in a limiting
sense, and the scope of the present invention is defined only by
the appended claims.
[0069] To carry out the objects and principles of the present
invention, an automatic critical angle refractometer was used to
sense the shadow line on a photo sensor, such as an LSA, and,
therefore, resolve the critical angle of total reflection of a
contacting phase, placed on an optically transparent element with a
binding layer on it. The binding layer comprised the ligands with
specific affinity to the analyte in the contacting phase.
(Throughout this description the words "sample" and "contacting
phase" are used interchangeably). When the analyte bonded to the
binding layer, the optical density of the binding layer changed. In
several experimental trials, described in detail below, a critical
angle refractometer was used to detect changes in the optical
density of the binding layer occurring as a result of the binding
phenomena. The experimental results described below demonstrated
that small refractive index changes, occurring as a result of
binding between the binding layer and the sample analyte, could be
detected using the opto-electronic configuration of the present
Leica AR600 automatic refractometer, which provides refractive
index measurement over a relatively broad range of indices. The
results of the experimental trials proved that critical angle
refractometry can be successfully used to detect and monitor
changes in the optical density at the binding layer, which changes
are caused by binding interactions between the analyte and the
binding layer.
[0070] FIG. 7 shows a test assembly 24 for performing
refractometric measurements in the preferred embodiment of the
present invention. FIG. 7 shows the test assembly in slightly
exploded detail. Test assembly 24 includes a high index coupling
liquid 76, introduced directly to top surface 54 of prism 50 of
critical angle refractometer 22 (not shown), an optically
transparent element, such as disc 78, with a binding layer 51',
deposited on an interface 80 between disc 78 and the binding layer,
and a sealing 0-ring 82 interposed between flow cell 28 and
interface 80. In this embodiment prism 50 is a sapphire prism with
the refractive index of about 1.7.
[0071] Binding layer 51' comprises ligands 53 immobilized on
interface 80. Ligands 53 may or may not have specific binding
affinity to the sample analyte contained in a contacting phase 59.
In the preferred embodiment of the present invention, contacting
phase 59 is a liquid phase, delivered through flow cell 28 to
contact binding layer 51'. After the contact, the sample analyte in
phase 59 contacts ligands 53 and binds to the ligands, provided
that the analyte and the ligands have specific affinity to each
other, allowing the binding phenomena to occur. An example of
binding layer 51' is an antibody matrix immobilized on interface
80. Contacting phase 59, such as, for example, an antigen solution
for interaction with the antibody matrix of layer 51', is delivered
by flow cell 28, and the binding interaction is then monitored by
means of critical angle refractometry.
[0072] Flow cell 28 can be a conventional flow cell. In one of the
embodiments of the present invention, a flow cell capable of
providing a flow rate of about 1 ml/minute, such as that available
from Leica Microsystems Inc. under Catalog No. 10610, covers a
substantial portion of interface 80. A suitable coupling liquid 76
is high refractive index oil, preferably 1.63 refractive index oil.
Transparent disc 78 can be formed of a material that is optically
transparent to the incident light, such as, for example, glass,
plastic, or other optically transparent materials with a suitable
index of refraction. In the described embodiment, the wavelength of
incident light is 589 nm. In the preferred embodiment, disc 78 has
a refractive index greater than 1.52 at 20.degree. C. Such
materials are, for example, glass, polystyrene, or polycarbonate. A
suitable thickness for discs 78 used in experimental trials was
0.17 nm.
[0073] Another embodiment of a flow cell/transparent disc test
assembly is illustrated in FIGS. 7a-7e. In that embodiment a base
200 of the flow cell is placed on a plate 202, which plate has an
opening 204 exposing top surface 54 of prism 50 (prism not shown),
as shown in FIG. 7a. Base 200 can be attached to plate 202 by any
convenient means, including simply placing the base on the plate,
or using screws or other equivalent means to fasten the base to the
plate through openings 206, as shown in FIG. 7a. A drop of coupling
liquid 76 (shown in FIG. 7) is put on top surface 54 either
manually or by bringing liquid 76 to top surface 54 by dropping the
liquid into a first plate opening 208. The liquid then travels
through a first tube 210 connected to a plate opening 208 and
reaches surface 54. If an excess amount of coupling liquid 76 is
brought to top surface 54, a second tube 212 drains the excess
liquid from top surface 54 to a second plate opening 214, as
illustrated in FIG. 7e.
[0074] FIG. 7b shows base 200 and plate 202 with an optically
transparent slide 218 (similar to transparent disc 78 described
above) placed on plate 202. Slide 218 is disposed above top surface
54 of the prism and inside a frame 207 in base 200 suitable for
receiving the slide. Frame 207 defines the location of slide 218
relative to top surface 54 of the prism. In FIG. 7b frame 207 is
rectangularly shaped to receive a rectangular slide 218 and to
partially surround the slide. As can be seen in FIG. 7e, in one of
the embodiments slide 218 comprises an optically transparent area
236 with a binding layer attached to it, and a frosted area 237 for
trapping and diffusing light illuminating area 237. Area 237 can be
etched and mechanically reduced with an abrasive to produce the
frosted finish.
[0075] FIG. 7c illustrates a cap 240 of the flow cell with tubes
226 and 228 for circulating a contacting phase through the flow
cell. Cap 240 comprises a cap frame 230 with holes 222. One or more
threaded studs 216 protruding from base 200 pass through the holes
222 in cap frame 230 which is then secured in place preferably by
knurled nuts 234, as illustrated in FIG. 7d. Tubes 226 and 228 are
coupled to the flow cell via a top portion 232 fitting into cap
frame 230, as shown in FIGS. 7c, 7d, and 7e. Top portion 232 can be
attached to cap frame 230 either permanently or removably. Tubes
226 and 228 deliver contacting phase 59 (shown in FIG. 7) to slide
218. The contacting phase enters the flow cell through one of the
tubes 226 or 228, flows over the slide and exits the flow cell
through the other tube. It is contemplated that top portion 232 can
comprise a temperature sensor or any other sensor measuring various
properties of the fluids circulating in the flow cell. Such a
sensor would be disposed on the surface of top portion 232 normally
in contact with the contacting phase during the operation of the
refractometer.
[0076] A particular geometry and design of slide 218 used in the
flow cell assembly described with regard to FIGS. 7a-7e are shown
in more detail in FIGS. 17a-17e. Slide 218 is shown to have an
upper surface 250 with a first raised portion 252 (FIG. 17a ).
Upper surface 250 has a frosted finish, diffusing or trapping
unwanted light and preventing it from entering the sensing system
and being detected by the light sensor. Frosted finish can be
achieved by chemical or mechanical etching or any other means
contemplated by a particular application. First raised portion 252
is not etched and remains optically transparent to light. A
cross-sectional side view of slide 218 shown in FIG. 17e
illustrates a raised portion 252 having a sensing surface 253 onto
which binding layer 51' is attached. In one of the embodiments
illustrated in FIGS. 17a-17d first raised portion 252 is shaped as
an oval having a longitudinal axis X. A gasket 220, shown in FIG.
7b, is usually placed over first raised portion 252 to seal the
area of circulation of the contacting phase during the measurement
time. Gasket 220 In FIG. 7b is oval shaped to parallel the shape of
first raised area 252 in FIG. 17a.
[0077] A lower surface 254 of slide 218 in FIG. 17b is similar to
upper surface 250, the frosted finish of lower surface 253 prevents
unwanted light from entering the sensing system of the
refractometer. A second raised portion 256 is not etched and
remains optically transparent to light. Second raised portion in
one of the embodiments is tear-drop shaped with a longitudinal axis
Y perpendicular to axis X. A third raised portion is also
transparent to light and comprises a rim 258 which contacts plate
202 (shown in FIG. 7a) when the slide is placed on the plate.
Coupling liquid 76 delivered to top surface 54 of the prism
contacts second raised portion 256 and couples slide 218 to top
surface 54, as illustrated in FIG. 7b.
[0078] As follows from FIGS. 17c-17d, an area of transparent
overlap 260 defined by raised portions 252 and 256 is the area
where the light traveling through slide 218 and illuminating
surface 253 will be reflected or transmitted, indicating the
presence or absence of the binding reaction between binding layer
51' and the analyte in the contacting phase. As shown in FIG. 17d,
the oval-shaped portion 252 extends beyond the overlap area 260
from a first end 262 to a second end 264. A contacting phase in the
flow cell usually first contacts slide 218 either at first end 262
or second end 264. By the time the contacting phase travels from
either first end 262 or second end 264 and to overlap area 260, any
turbulence in the contacting phase is reduced and the flow of the
contacting phase becomes laminary, improving the precision of the
measurements of the binding reaction on the sending surface.
[0079] As illustrated in FIGS. 17a and 17d, the present invention
contemplates that surface 250 of slide 218 comprises a code area
270, which contains an indicium or an embedded chip. The indicium
contained in code area 270 can be a readable optical pattern
providing information about a particular binding layer attached to
the slide, refractive index of the material of the slide, or a
particular slide in any desired way. The indicium also can be used
to ascertain that the slide is correctly inserted into the flow
cell before the beginning of the measurement session. If code area
270 contains a chip, the chip can be responsive to certain energy
(such as, for example, radio frequency energy) with a distinctive
coded signal. In response to the distinctive coded signal the chip
will provide information about a particular slide, the binding
layer attached to the sensing surface, the orientation of the slide
and any other desired information. Code area 270 can be located on
any surface of the slide or inside the slide, depending on a
particular design of the slide, the flow cell and the
refractometer.
[0080] It should be noted that other geometries and designs of
slide 218 with a binding layer on it can be used without departing
from the scopes of the claims of the present invention.
[0081] It is noted that the use of transparent disc 78 for
supporting the binding layer is preferred, but not necessary in all
instances for practicing the present invention. This is so,
because, for example, binding layer 51' can be immobilized directly
on prism 50 without disc 78. In that case the sample analyte in
contacting phase 59 contacts ligands 53 immobilized directly on
prism 50. In such an arrangement, as well as other possible
arrangements, as long as the condition of total internal reflection
is satisfied, refractometric measurements of binding phenomena can
be performed.
[0082] Indeed, various arrangements can be used for fulfilling the
condition of total internal reflection at interface 80 in order to
resolve the critical angle. For example, a photo sensor can be
positioned to sense either the portion of light, which was totally
internally reflected at the interface (as in the preferred
embodiment of the invention), or the portion of light transmitted
through the interface into the binding layer. In both cases the
position of the shadow line on the sensor will be indicative of the
refractive index of the binding layer and, therefore, of the
binding phenomena taking place at the binding layer. Additionally,
interface 80 can be illuminated with either transmitted or
reflected light. The condition for total internal reflection can be
also met, when the interface is illuminated through any optical
system, not necessarily the optical system, which includes a prism
directing light onto the interface. Such optical systems as, for
example, lens or mirror arrangements directing the light onto the
interface can be successfully used in the present invention. As
long as the light incident on interface 80 illuminates the
interface at the angles, satisfying the total internal reflection
condition, the refractometric measurements of the binding phenomena
can be made.
[0083] By way of example, but not limitation, various binding
layers, suitable for use with the critical angle refractometer of
the present invention, can comprise the following ligands,
immobilized on transparent disc 78. The first example is a
carboxylated dextran coated glass disc. In that example, a layer of
dextran is first chemically bound to the surface of transparent
disc 78 made of glass. After the dextran has been immobilized on
disc 78, it can be modified with one of the several different
chemicals containing a carboxyl group as a terminal end of the
dextran molecules. The modification step provides carboxymethyl
groups, which can be used to bind such ligands as proteins by
well-known EDC/NHS chemistry. Examples of the chemicals used at the
modification step include chloroacetic, bromoacetic, and
6-bromohexanoic acids. After the dextran has been modified, direct
immobilization of such binding layers as proteins to the carboxyl
group can be accomplished, further modification of the dextran can
be performed to attach to it a terminal amine group using the known
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodi- amide)
chemistries.
[0084] Another example of a binding layer includes silane molecules
covalently bound to the --Si--OH groups on the surface of disc 78
made of silica. Depending upon the functional groups located at the
terminal end of the bound silane molecules, direct immobilization
of proteins can be accomplished. Alternatively, further surface
modifications, i.e. attachment of molecules to the silane
molecules, can make other functional groups available for protein
(ligand) immobilization. Examples of commercially available silane
molecules are .gamma.-glycidoxypropyl triethoxysilane and
3-aminopropyl triethoxysilane. The functional groups at the
terminal ends of these silane molecules (the ends where
immobilization of a binding layer occurs) are hydroxyl and amine
groups, respectively. Alternatively, bifunctional groups, such as
gluteraldehyde, may be attached to the silane, and then the ligands
are attached to the group. The bifunctional groups are used as
spacer arms in order to minimize steric effects and provide access
to active sites of the ligands.
[0085] To obtain an aviden/streptaviden coated glass disc, aviden
or streptaviden is bound to the glass surface of disc 78, using
chemistries similar to those described in the silanization
procedure. Once silane is bound to the glass surface, then
aviden/streptaviden is attached to a bifunctional spacer arm. Since
the bioten-aviden interaction is characterized by one of the
highest available affinities, the surface with attached
aviden/streptaviden possesses an extremely high affinity for
biotin. Biotinylated proteins can then be easily bound to the
coated glass by merely bringing biotinylated proteins into contact
with aviden.
[0086] In another example of binding chemistry, biotin molecules
are bound to the surface of disc 78 made of glass, creating the
initial biotinylated glass surface. The binding is accomplished by
using chemistries similar to those described in the silanization
procedure, resulting in biotinylated glass. The aviden/streptavlaen
molecules are then selectively adsorpted on the biotinylated glass
surface, thus creating a surface with high affinity for biotin
molecules, similar to the surface of the aviden coated glass.
Biotinylated proteins can then be attached to the surface. In this
example, the surface can be regenerated without destroying the
initial biotinylated glass surface. Alternatively,
aviden/streptaviden/neutraviden conjugated proteins may be directly
coupled to the biotinylated glass.
[0087] When transparent disc 78 is made of plastic, several binding
chemistry techniques are available to immobilize various ligands on
the plastic surface. One of the techniques is a passive adsorption
of proteins to a hydrophobic plastic material with no modification
or activation of the proteins. The preferred hydrophobic material
is polystyrene. In that technique electrostatical attraction causes
positive charges on a protein "stick" to the negatively charged
plastic. By controlling such parameters as pH, ionic strength, and
period of incubation, binding of the ligands (proteins) can be
accomplished. The period of incubation here is the length of time
during which the solution containing the protein is left in contact
with the disc surface.
[0088] Another available technique involves, as an initial step,
binding glutaraldehyde to the plastic surface, leaving the aldehyde
group at the terminal end. The aldehyde group is then used to
immobilize a protein. Such a technique usually results in increased
amount of immobilized protein. Yet another immobilization technique
involving plastic surfaces is the technique of using
photo-activated cross-linkers. In that technique immobilized
enzymes are conjugated with cross-linker molecules and then bound
to the plastic surface by exposure to UV light.
[0089] It is also contemplated in the present invention that a self
assembled lipid monolayer on a clear glass or plastic surface can
be used to immobilize membrane bound proteins or other ligands with
affinity for the hydrophobic surfaces. Overall, the suitable
binding layers can be carboxymethylated dextran, aldehyde activated
dextran, hydrazide activated dextran, silanated surfaces, silanized
surfaces, silane, aviden, streptaviden, neutraviden, biotinyl,
bifunctional spacer arms, self assembled monolayers, lipids and
unchanged or uncoated surface of glass or plastic. The suitable
analytes can be antigens, proteins, glycoproteins, vitamins,
microbes, pieces of microbes including bacteria and bacterial
fragments, viruses, pieces of viral material, lipids,
carbohydrates, toxins, DNA, RNA, DNA and RNA analogs, pathogenic
organic molecules, anti-bacterial and anti-viral organic molecules
and their analogs, therapeutic agents and drugs.
[0090] In experimental trials described in detail below,
refractometer 22 was used in an experimantal set up illustrated in
FIG. 3b. Shown in FIG. 3b is an experimantal set up formed in
accordance with the present invention and identified broadly as
reference numeral 20. Set up 20 generally comprises automatic
refractometer 22 having test assembly 24 thereon for measuring a
refractive index, a reservoir 26 for storing a fluid contacting
phase, flow cell 28 defining a well 29 (see FIG. 10) adjacent test
assembly 24, a pump 25 and flexible tubes 27 for delivering the
contacting phase from reservoir 26 to flow cell 28, and a personal
computer 30 connected for serial communication with refractometer
22 for controlling measurement functions of the refractometer, and
for processing and storing measurement data received from the
refractometer.
[0091] Refractometer 22 further includes an RS-232 serial port, not
shown, for data linking by way of a standard serial cable 42 to a
peripheral device, most commonly personal computer 30 having its
own serial communications port (COM1 or COM2). Communication
between refractometer 22 and computer 30 can be controlled by any
terminal communication software program running on the computer.
However, the terminal program, which comes with Microsoft Windows
3.11 and the hyperterminal program, which comes with Microsoft
Windows 95, are known to enable data communication between
refractometer 22 and computer 30 for extracting real time
measurement data according to methodology of the present invention.
The recommended communications port setup for the terminal program
is baud rate--19200, data bits--8, stop bits--1, parity--none, flow
control--XON/XOFF. Under the menu option 5 "Settings", then option
"Text Transfers", the option "Standard Flow Control" should be
chosen. It is helpful to save these settings for future use. As
will be understood more fully by reference to the Owner's Manual
and supporting documentation for the Leica AR600 automatic
refractometer, the refractometer may be controlled remotely from
computer 30 through user input and sending of various control
codes. While the apparatus of the present invention is generally
described herein including automatic refractometer 22 linked to
personal computer 30, it is of course possible to provide
preprogrammed software and memory within refractometer 22 itself,
which enables the instrument to continually perform and record
readings at regular chosen intervals for various sensing
applications.
[0092] It is important to note that in the commercially available
Leica AR600 automatic refractometers, a crossover at cell #100 of
the LSA corresponds to a refractive index of about 1.3 at
20.degree. C., while a crossover at cell # 2450 corresponds to a
refractive index of about 1.52 at the same temperature. Since most
binding phenomena result in changes of the refractive indices
within the range from about 1.3 to about 1.4, the available broad
range of refractive index measurements in the configuration of
Leica AR600 was successfully used.
[0093] By way of overview, seven experiments described herein
involved two general stages: first, preparation of a supporting
surface and immobilization of a binding layer thereon, and second,
continuous measurements of refractive index changes at the
interface between the glass and the binding layer by critical angle
refractometry. The measurements were taken after additions of a
non-binding solution to the contacting phase and during the binding
reaction between the sample analyte and the binding layer.
[0094] In trials one and two, the mass of the flowing contacting
phase was changed by additions of sucrose and Bovine Serum Albumin.
The additions were made in order to determine feasibility of
measuring refractive index changes at the surface of a glass slide
coupled to the prism of the Leica AR600 critical angle
refractometer.
[0095] In the third and fourth trials, antibody was immobilized by
covalent bonding to silanated glass discs, which contained a
proprietary spacer arm terminating in an aldehyde group.
Measurements were taken for five successively increasing
concentrations of antigen added over time in each trial.
[0096] In trial five, Neutraviden conjugated Goat Anti-Mouse IgG
antibody was adsorbed onto a biotinylated glass surface. In trials
six and seven the experiment included the reaction of immobilized
Goat Anti-Mouse Antibody with Sheep IgG as a control solution and
the specific binding antigen Mouse IgG. The recorded measurement
data from experimental trials one through seven are provided in the
corresponding drawing figures.
[0097] Preparation of transparent discs 78 involved silanyzation
and then periodate oxidation of the discs. While silanated glass
slides are available commercially and may be useful in practicing
the present invention, silanyzation of plano-plano glass plates of
a suitable refractive index, which is greater than the expected
refractive index of a contacting phase was performed to ensure high
quality in trials one and two. The transparent discs 78 used in the
experiments were initially cleaned and hydroxylated by consecutive
immersion in concentrated sulfuric acid, distilled water, ethanol,
and acetone. Five hundred grams of sulfuric acid were acquired from
Sigma-Aldrich Chemicals, product number S-1526, and was of ACS
Reagent Grade, 95.7% pure. Transparent discs 78 were immersed once
for ten minutes in the sulfuric acid, and then immersed in
distilled water three times, for ten minutes each time. The ethanol
was acquired from Sigma-Aldrich Chemicals, product number 27,074-1,
and was reagent, denatured, HPLC Grade. Transparent discs 78 were
immersed in the ethanol twice for ten minutes per each immersion.
The acetone was also obtained from Sigma-Aldrich Chemicals and was
99.9% pure, ACS Reagent Grade. The transparent discs were immersed
twice for ten-minute periods in the acetone. Various immersions can
be carried out by supporting transparent discs 78 on wire support
hooks each of which is formed from a single strand of copper wire.
Several of these hooks, each having a transparent disc 78 mounted
thereon by bending the wire around the disc, were hung from a bar
set across the top of a reagent container. As an alternative to
individual immersion hooks, one or more racks having an array of
regularly spaced open wells for holding transparent discs 78 could
be machined specifically for immersion purposes.
[0098] Once the transparent discs 78 have been cleaned, they must
be activated with 3-aminopropyl,tri-ethoxy silane, hereinafter
referred to as APTS. An APTS molecule contains a silicon atom
bonded to three ethyl groups via an oxygen atom (tri-ethoxy
silane). This portion of the molecule bonds directly to interface
80 of transparent disc 78 during a condensation reaction involving
the hydroxy groups on interface 80, and the free protons in the
aqueous environment. To perform this reaction for a batch of twenty
discs, a 10% wt/vol fresh aqueous solution of APTS is produced by
mixing 2.5 ml of APTS with 25 ml of distilled water. This solution
was titrated to a pH of 5.0 using Glacial Acetic Acid, obtained
from SigmaAldrich Chemicals, product number A-0808, and an
electronic pH meter. The reaction of transparent discs 78 with the
coupling agent APTS was controlled at 80.degree. C. for three
hours.
[0099] FIG. 14 illustrates an arrangement used for restricting
chemical activation to only one side of each transparent disc 78.
Transparent disc 78 was placed on a narrow, sturdy surface, such as
the surface of a transparent slide 84, with the sample surface to
be activated facing upwards as shown. A rubber 0-ring 86 having an
inner diameter of 8-9 mm is placed on top of transparent disc 78.
The size of 0-ring 86 is chosen to be the same as or slightly
larger than the size of 0-ring 86 placed between flow cell 28 and
transparent disc 78 during the actual refractometric readings to
ensure that all of the sample surface exposed to the sample analyte
during the refractometric measurements will be activated. A piece
of tubing 88 having an inner diameter corresponding to that of
0-ring 86 and a height no greater than 25 mm is placed on top on
0-ring 86. As will be appreciated, 0-ring 86 creates a fluid-tight
seal between transparent disc 78 and tube 88. Tube 88 includes a
pair of diametrically opposed through-holes at an elevated portion
thereof for receiving a sturdy dowel 90 having exposed end portions
90a and 90b. The assembled components are releasably held together
by a continuous elastic band 92 looped around one exposed end 90a
or 90b of dowel 90, then under slide 84 and around the other
exposed end of dowel 90.
[0100] After the reaction of the APTS solution with the sample
surface 80 of transparent disc 78 is completed, the assembly shown
in FIG. 14 was disassembled and transparent discs 78 were heated
for two hours at 120C., then cooled to room temperature. The cooled
transparent discs were then soaked in 5% wt/vol glutaraldehyde
solution in phosphate buffer. The pH of the phosphate buffer was
6.8. For a batch of twenty to thirty transparent discs, mixing 10 g
of glutaraldehyde with 200 ml of phosphate buffer provided a
sufficient amount of solution. The transparent discs were soaked
for ninety minutes at room temperature (22' C.), followed by two
immersions in distilled water for ten minutes each immersion. After
the water immersions, the single side activation assembly shown in
FIG. 14 was reassembled and a small amount of the antibody was
pipetted into the tubing well and allowed to react for twenty-four
hours at 40 C. to test coating efficiency. Following the antibody
reaction, transparent discs 78 were washed with phosphate buffer
and stored in isotonic saline at 4' C. The method of silanyzation
is well known, and reference can be made to the publication
Immobilized Affinity Ligand Techniques by Greg T. Hermanson, A.
Krishna Mallia, and Paul K. Smith, Academic Press, 1992, pages
12-14.
[0101] To carry out the periodate oxidation step, 600 .mu.l of at
least 1 mg/ml antibody solution was extracted and placed in a
labeled vial, and 0.06 g of sodium meta-periodate was dissolved in
10 ml of distilled water. Periodate solution was then combined with
antibody solution by mixing 300 .mu.l of periodate solution with
the antibody solution in the labeled vial, and allowing the
combination to react in the dark for thirty minutes to produce
aldehyde groups from the carbohydrates. The aforementioned
publication Immobilized Affinity Ligand Techniques provides
protocol for periodate oxidation of a support matrix at page 75
thereof. The next step was to couple the antibody to interface 80
of silanated discs 78. Protocol for this step may be found
Immobilized Affinity Ligand Techniques starting at page 223.
[0102] In standard refractometric procedures refractometer 22 is
initiated in air with nothing on top of prism 50. In that case all
light incident on the interface between the prism and the air is
reflected, because the prism's index of refraction is usually much
higher than that of the air. Therefore, when the refractometer is
initiated in air, the sensor detects all the light, and, therefore,
there is no shadow line formed on the sensor. Accordingly, the
refractometer puts out a reading, which looks like reference curve
100 on FIG. 5 and FIG. 6. Also, under the standard operating
conditions, a sample is usually placed on top of the prism with no
ligands immobilized on top of the prism. With a sample placed on
top of the prism, the critical angle phenomenon is satisfied, and
the light incident on the interface becomes partially reflected and
partially transmitted, forming the shadow line on the sensor. The
refractometer, therefore, produces a reading looking like sample
curve 110 in FIG. 5.
[0103] Since in the preferred embodiment of the present invention
the refractometer is used to sense and monitor interactions between
various ligands with transparent disc 78 placed on top of the prism
and immobilized binding layer 51' on the disc, initiating the
refractometer in air is undesirable. Therefore, in the preferred
embodiment of the invention the initialization procedure is
performed not in air, but in water. To initialize the refractometer
in water, a drop of a coupling liquid, such as oil, was deposited
on top of prism 50, then an optically transparent disc was placed
on top of the prism. A flow of water was flown over the disc, and
the refractometer was allowed to initialize in water, producing the
reading such as a water reference curve 130 in FIG. 6. It is noted
that it is also possible to initialize the refractometer with the
disc on the prism by flowing a low concentration PBS
(physiologically buffered saline) over the disc, instead of flowing
water. The suitable PBS has a pH of about 7.4 and comprises, for
example, 0.14 M NaCl with 0.01 M or 0.1 M phosphate buffer with pH
of about 7.4.
[0104] If the refractometer was initialized with water or a diluted
PBS, as described above, then its calibration procedure can not be
performed with water. In that case, a flow of a standard PBS is
used to calibrate the instrument, producing a reading, looking like
a PBS calibration curve 140 in FIG. 6. Once the refractometer has
been initiated and calibrated in accordance with the
above-described procedures, it is ready for operation.
[0105] During the preparation stage of the experimental trials
standard silica glass microscope slides were purchased from Fisher
Scientific. Xenobind glass slides were purchased from Xenopore, X,
N.J., USA. Xenobind slides are made of silanized glass with a
proprietary spacer arm covalently bound to the glass and with an
aldehyde group at the terminal end. Biotinylated glass slides were
prepared according to the method of Swaisgood et. al, 1997: glass
slides were cleaned in 1:4 v/v nitric acid (available from Sigma,
St. Louis, Mo., USA) at 95.degree. C. for 1 hour, followed by
rinsing with distilled water. Slides were then immersed in 10% v/v
3-aminopropyltriethoxysilane--HCl (pH 4.0), incubated at 70.degree.
C. for 3 hours and dried at 110.degree. C. overnight, immersed in 5
mg/nl NHS-LC-Biotin (sulfosuccinimidyl 6-(biotinamido) hexanoate
(available from Pierce, Rockford, Ill.) in 50 mm sodium bicarbonate
(pH 8.5) for 2 hours at 4.degree. C., washed and stored in 50 mm
sodium phosphate buffer (pH 6.0) containing 0.02% NaN3. Prior to
the start of the experimental trials, slides were cut into 100
mm.sup.2 square sections. PBS, used in all of the described
experimental trials as a control solution for calibration purposes,
contained 10 mm of sodium phosphate, 0.138 m of sodium chloride,
2.7 mm of potassium chloride and 0.02% Tween-20. The purpose of the
detergent was to minimize non-specific binding.
[0106] Each of the Rabbit Anti-Sheep and Goat Anti-Mouse Antibodies
was then immobilized on a Xenobind slide as follows: 40 ml of 1
mg/ml antibody was diluted with 400 ml 0.15M NaCl, 0.1M Sodium
Bicarbonate (pH 9.1). The diluted antibody was spread over the
surface of the Xenobind slide. The slides with the immobolized
antibodies were then placed in a humidified sealed container
overnight in darkness. After that the slides were rinsed with
distilled water, dried under a nitrogen stream and immediately used
for experimental trials.
[0107] For experiments with NeutrAviden conjugated antibodies, the
antibody was immobilized on a biotinylated glass slide as follows:
40 ml of 1 mg/ml antibody was diluted with 400 ml of PBS containing
0.02%Tween. Then the diluted antibody was spread onto the slide and
allowed to incubate for 3 hours at room temperature in a
humidified, sealed, container. The slide was then rinsed with
distilled water, dried under a nitrogen stream and immediately used
for experimental trials. Alternately, NeutrAviden conjugated
antibodies were immobilized on the glass slide by adding them to
the mobile phase during the course of the trial.
[0108] All experimental trials were conducted according to the
following preferred procedure, in which reference is made to the
test assembly shown in FIG. 7. A small drop of 1.63 refractive
index coupling oil (corresponding to coupling liquid 76 in FIG. 7)
was placed on the top of prism 50. The glass slide, corresponding
to reference numeral 78 in FIG. 7, was carefully placed on top of
the drop of oil to avoid trapping air bubbles between the slide and
prism 50. Great care was also taken to be sure the oil did not
contact interface 80 with binding layer 51' containing immobilized
antibodies (ligands 53 in FIG. 7). The flow cell assembly was
completed as described earlier in connection with FIG. 7. PBS
containing Tween was then circulated over the slide at 1-2 ml/min
for 30 min prior to the start of each experimental trial. The Leica
AR600 refractometer was initiated after flowing distilled deionized
water over the slide for 3 minutes. Initialization of the
refractometer with distilled deionized water provided a reference
line of the linear scanned array. The reference line was later used
by the software to determine shifts in the shadow line indicative
of the critical angle of total reflection. The solution was then
switched to PBS with Tween for 5 minutes to calibrate the
refractometer. Once calibrated, the refractometer was set to read
refractive indices at a series of pre-set intervals. For the
experimental trials described below, 124 scans of the linear
scanned array were taken for each reading.
[0109] Typically, PBS was flown over the slide surface until a
stable reference line was achieved. Contacting phase 59, containing
the control protein or an antigen as a sample analyte, was then
added to the reservoir (reference numeral 26 in FIG. 3b ) and the
refractive index of the binding layer 51' was monitored for about
10 to 30 minutes. The control solution was run to distinguish
non-specific binding from true antigen/antibody binding
interactions. In addition, increasing the antigen concentration in
the reservoir provided information about the dependence of the rate
of binding interactions on the flow rate and antigen
concentration.
[0110] The first experimental trial was conducted to show that a
critical angle refractometer with the preferred test assembly,
illustrated in FIG. 7, could detect changes in the refractive index
of a bulk solution, contacting glass slide 78 with no binding layer
51' on the slide. At the beginning of the experiment, the
refractometer was initiated with distilled deionized water and
calibrated with PBS, as described above. During the first
experimental trial the change in the refractive index was monitored
as a function of time for successive additions of sucrose to the
recirculating PBS buffer. The observed changes in the refractive
index, shown in FIG. 8, were as expected from the well-known
relationship between the concentration of sucrose and the
refractive index. It was also noted that the changes in the
refractive index were immediate, which, again, was an expected
observation due to a high flow rate (>1.0 ml/min) of the
contacting phase and a low flow cell volume (<30 ml), resulting
in minimal dilution effects.
[0111] The second experimental trial was similar to the first one,
except that a large mass of added protein Bovine Serum Albumin
(BSA) was used to observe the change in the refractive index.
Similarly to the first trial, the change in the refractive index,
illustrated in FIG. 9, was immediate and of the expected magnitude
for the added mass of BSA. The BSA trial also demonstrated that the
effects of non-specific binding to the binding layer were
minimal.
[0112] The first and second experimental trials demonstrated that
the described configuration of the AR600 refractometer could detect
refractive index changes in a contact phase flowing over a glass
slide, coupled by a high index oil to the prism of AR600.
[0113] The next four experimental trials (third through sixth) were
conducted to show that the same configuration of the critical angle
refractometer can detect changes in the refractive index at a
binding layer, deposited on the slide, in contact with a sample
analyte in a contacting phase. In these trials, the ligands in the
binding layer were immobilized antibodies, the contacting phase was
PBS, the sample analyte was the antigen with specific affinity to
the antibodies. A control solution containing PBS with a
non-specific antigen or protein, such as BSA, was also used to make
sure that the subsequent observed index changes were due to
specific antigen/antibody binding interaction, and not due to
non-specific antigen/antibody interaction.
[0114] The measurements taken during the third experimental trial,
shown in FIG. 10a, represent the refractive index changes at the
binding layer of Rabbit Anti-Sheep Antibody immobilized on a
Xenobind glass slide. As seen in FIG. 10a, when a stable reference
line was established, Mouse IgG was added to the reservoir as a
control solution, after which the refractive index was monitored
for 45 minutes. Similarly to the observations in the first and
second experimental trials, the addition of the non-binding control
Mouse IgG solution resulted in the immediate change in the
refractive index, due to a high salt concentration in the buffer
containing the Mouse IgG. After the immediate change, the
refractive index remained almost unchanged for 45 minutes.
Subsequent volumes of 50 g/ml Sheep IgG Antigen, having specific
affinity to the antibody, were then added to the reservoir every 45
minutes. No immediate jump in the refractive index was observed,
due to the fact that the buffer containing the Sheep IgG Antigen
was identical to the circulating contacting solution. As can be
seen in FIG. 10a, gradual changes in the refractive index were
detected each time a quantity of the antigen was added to the
contacting phase, which is an indication of the specific binding of
the antigen to the immobilized antibody. The data in FIG. 10b, fit
by using linear regression, also illustrate that the rate of change
in the refractive index increases as the concentration of the
antigen increases.
[0115] In the fourth experimental trial, Goat Anti-Mouse IgG
Antibody was immobilized on a Xenobind slide, as described above.
Similarly to the results of the third experimental trial, the
refractometer detected changes in the refractive index each time a
quantity of antigen was added. As can be seen in FIG. 11a, after
the reference line was established, 50 g/ml of non-binding Sheep
IgG was added to the reservoir as a control solution. No change in
the refractive index was observed with the addition of the
non-antigenic non-binding Sheep IgG molecules. Also, no change in
the refractive index was observed with large additions of Bovine
Serum Albumin (the BSA data are not shown). The Mouse IgG antigen,
having specific affinity to the Anti-Mouse IgG antibody, was then
added to the reservoir in 10 g/ml increments at approximately 25
minute intervals. As shown in FIG. 11a, the changes in the
refractive index were quite large and gradual, as opposed to the
immediate changes in the index caused by the mass addition to the
contacting phase. Similarly to the data in FIG. 10b, the data in
FIG. 11b, fit with linear regression, represent the changes in the
refractive index detected by the refractometer during the
measurements of the reference line, control solution and antigen
additions. As with additions of the Sheep IgG Antigen in the third
trial, the rate of change of the refractive index in FIG. 11b
increases with the increase of the concentration of the Mouse IgG
antigen. Evidently, the gradual increase is caused by binding, and
not by the mass addition to the contacting phase. The increase is
also of a much greater magnitude than what can be attributed to the
mass addition. Therefore, the changes in the index, shown in FIGS.
14a and 14b, were caused by the antigen/antibody binding
interactions, detected by the refractometer.
[0116] The fifth experimental trial was conducted to demonstrate
the ability of the refractometer to sense and monitor binding of
the NeutrAviden conjugated Goat Anti-Mouse antibody to a
biotinylated glass slide. At illustrated in FIG. 12, after the
reference line was established, 15 g/ml of the NeutrAviden
Conjugated Goat Anti-Mouse Antibody was added to the reservoir.
After approximately 40 minutes and an index change of 0.002, the
index stopped changing, which is indicative of the saturation of
the antigen/antibody binding reaction. Since the index change of
0.001 corresponds to approximately 1 ng/mm.sup.2 of the immobilized
antibody, the total antibody/antigen bound to the surface of the
slide is 2 ng/mm.sup.2 or 200 ng/cm.sup.2. Such a result is well
within the range, expected for these types of experiments.
[0117] In the sixth experimental trial, the NeutrAviden conjugated
Goat-Anti Mouse antibody was immobilized on a biotinylated glass
slide. The immobilization step was performed by spreading the
diluted antibody solution on the surface of the slide and
incubating for 3 hours prior to assembling the slide into the
refractometer to prevent non-specific binding of the NeutrAviden
conjugated Goat Anti-Mouse antibody to the tubing and the
reservoir. (Subsequent experiments with the antigen were much more
reproducible, when the entire flow cell arrangement was not
pre-exposed to the NeutrAviden conjugated antibody). FIG. 13a
illustrates the changes in the refractive index at the binding
layer of the NeutrAviden conjugated Goat Anti-Mouse antibody
immobilized on the slide during the course of the experimental
trial. Similarly to the previous experiments, the PBS reference
line was allowed to stabilize and the control non-binding Sheep IgG
solution was added in large excess. As can be seen in FIG. 13a, two
additions of 10 g/ml of the Sheep IgG control solution did not
result on any change of the refractive index. Alternately, a BSA
control solution was added. (The BSA data are not shown). The
control solutions were circulated to make sure that the subsequent
observed index changes were due to specific antigen/antibody
binding interaction, and not due to non-specific antigen/antibody
interaction. Additions of the Mouse IgG Antigen were then made to
the reservoir in the presence of either the IgG or BSA control
solution, or a combination of both. 0.02% Tween 20 detergent in the
PBS was also present to minimize non-specific binding. As can be
seen in FIG. 13a, the presence or absence of the control solutions
in the contacting phase had little or no effect on the refractive
index. Only when 10 g/ml and then 20 g/ml of the Mouse IgG Antigen
were added to the reservoir did the gradual changes in the
refractive index occur. Such changes, again, are attributed to the
specific antigen/antibody binding, detected and monitored by the
refractometer. FIG. 13b provides the fitted data, illustrating the
refracting index changes, corresponding to the addition of the
control Sheep IgG solution and two additions of the antigen to the
reservoir. Again, the observed changes occurred due to the
antigen/antibody binding interactions.
[0118] The seventh experimental trial, the results of which are
shown in FIGS. 16a-16d, was identical to the sixth trial, except
for the fact that 100 g/ml Mouse IgG antigen was added to the
solution to elicit a response. FIG. 16a shows changes in the
refractive index of the contacting phase after the addition of the
antigen. FIG. 16b illustrates intensity changes over the time
during which the seventh experimental trial was conducted. It can
be seen in FIG. 16b that the position of the shadow line relative
to the position of the shadow line during initiation of the
instrument ("G" and "B", respectively, in FIG. 16b ). FIG. 16c
shows that during the course of the experimental trial not only are
there changes in the position of the shadow line in response to the
addition of the Mouse IgG, but also there are changes in the
intensity of the response. FIG. 16d shows the position of the
shadow line at the beginning ("B") and at the end ("G") of the
experimental trial. These graphs illustrate how the position of the
intensity of the shadow line changes with the addition of the Mouse
IgG to the contacting phase and during the binding reaction between
the Mouse IgG and the binding layer immobilized on the slide.
[0119] The above described experiments demonstrated that critical
angle refractometry can be successfully used to detect and monitor
binding interactions between a sample analyte and a binding layer
by measuring changes in the refractive index at the binding layer.
Based on the experimental results, various embodiments of the
method and device of the present invention are described below by
way of example, and not limitation. It is intended that other
embodiments implementing the use of critical angle refractometry
for sensing and monitoring binding interactions between various
ligands fall within the scope and spirit of the present
invention.
[0120] As described above, in the schematic representation of the
optical measurement system of automatic refractometer Leica AR600
(FIG. 4) the light from source 48 travels along optical path 57 and
illuminates top surface 54 of prism 50 at various angles of
incidence. The portion of light, which is incident on surface 54 at
the angles greater than the critical angle, is reflected back and
sensed by LSA 44. The portion of light incident on surface 54 at
the angles smaller than the critical angle is transmitted into
sample 51 and escapes the LSA. The same principle can be used to
sense binding interactions in a different embodiment of the device,
in which light source 48 generates a collimated light beam incident
on top surface 54 of prism 50 or any other sensing surface at a
predetermined angle of incidence .alpha.. If, for example, the
binding layer is deposited directly on surface 54 of prism 50, then
interface 80 will be the interface between surface 54 and the
binding layer. Alternatively, if binding layer 51' is deposited on
transparent disc 78, then interface 80 will be the interface
between the disc and the binding layer.
[0121] For example, shown schematically in FIG. 15a is a light beam
92 generated by light source 48, which light beam 92 is incident on
interface 80 at angle .sub.1. The positions of light source 48 and
a sensor 98 in the embodiment will be predetermined. If a
particular analyte with specific affinity to binding layer 51' is
present in contacting phase 59, then the binding will occur, and
the refractive index n2 at the binding layer will increase, while
n1 remains unchanged. Since n2 increases, then, according to
Snell's law the critical angle of total reflection will increase.
Since in the described embodiment the angle of incidence.sub.1 is
fixed, choosing.sub.1 to be greater than or equal to the critical
angle when no analyte is bonded to the binding layer, but smaller
than the critical angle when the analyte is bonded to the layer,
will cause light beam 92 to be totally reflected at interface 80
with no analyte bonded, but to be transmitted through the
contacting phase when the analyte is bonded to the layer.
Therefore, when no analyte is bonded, reflected light beam 94 will
illuminate sensor 98. Once the analyte binds to the binding layer,
sensor 98 will not sense reflected light beam 94. Another sensor
(shown in dashed lines in FIG. 15a ) can be positioned to sense a
transmitted light beam 96, if a particular method and design of the
embodiment calls for sensing transmitted light. By sensing either
the presence of reflected beam 94 with sensor 98 or the presence of
transmitted beam 96 with sensor 98 positioned to detect the
transmitted beam (depicted by dashed lines in FIG. 15a ), the
presence or absence of binding interactions between the binding
layer and the analyte in the contacting phase can be determined. If
the embodiment uses one sensor, then the transition between sensing
and not sensing light will be indicative of binding. Sensor 98 can
be a simple photo sensing device, indicating whether beam 94 (or
beam 96) illuminates the sensing device, or an LSA, or any other
sensor capable of detecting light. Sensing binding interactions in
this embodiment of the invention can be used in devices aimed to
detect presence or absence of a particular substance in a sample.
An example of such a device is a "yes/no" test device, which is
able to indicate whether a particular type of ligands (protein,
antigen etc.) is present in the sample. Blood and urine samples,
for example, can be analyzed for presence of a particular substance
by a user at home or by laboratory personnel in a lab by using the
above described test device embodiment.
[0122] In yet another embodiment of the present invention, depicted
in FIG. 15b, a single light source 48 is allowed to move,
rotationally or translationally (shown by arrows in FIG. 15b ),
changing the trajectory of an incident collimated light beam 92 to
become either beam 92a or beam 92b, thereby altering the angle of
incidence of collimated light beam 92 (as depicted by dashed lines
in FIG. 15b). Similarly to the theory underlying the description of
the embodiment in FIG. 15a, when binding between the analyte and
binding layer 51' occurs, n2 increases, increasing the critical
angle of total reflection. If the original angle of incidence was
greater than or equal to the critical angle of total reflection
when no analyte was bonded to the binding layer, then sensor 98
senses reflected bean 94. When contacting phase 59 contains the
analyte reacting with the binding layer, then when binding occurs,
the angle of incidence no longer satisfies the condition of total
reflection. By moving or rotating source 48, a different angle
(such as .sub.1) greater then the critical angle is then selected,
so sensor 98 detects reflected beam 94b. In that embodiment, the
sensor can be an LSA, though it is not necessary. As in the
embodiment described with regard to FIG. 15a, sensing either
reflected beam 94 or transmitted beam 96 can be performed to detect
binding. The light source can be an LED.
[0123] Alternatively, instead of moving or rotating one light
source (source 48 in FIG. 15b), a plurality of fixed or movable
light sources 49 (FIG. 15c) can be used to direct light beams at
interface 80 at different angles of incidence. When the critical
angle increases due to binding between the analyte and binding
layer 51', a light beam from a different source 48' can be directed
to interface 80 at a greater angle of incidence .sub.1. If.sub.1 is
greater than the critical angle of total reflection when the
analyte is bonded to the binding layer, then sensor 98 will detect
binding by sensing reflected beam 94'. Sensor 98 can comprise one
sensor, such as an LSA, or a plurality of sensors, as shown in
dashed lines in FIG. 15c.
[0124] It is also contemplated that when a collimated light beams
illuminates interface 80, as described with regard to FIGS. 18b and
18c, instead of moving or rotating the light source, the incident
angle can be altered, and the condition of total internal
reflection can be satisfied, by moving or rotating the optically
transparent element on which the binding layer was immobilized.
[0125] As can be appreciated, the present invention encompasses an
apparatus and method for sensing and monitoring binding
interactions between various ligands by observing changes in the
refractive index over time attributed to binding. The refractive
index changes are observed by measuring changes in the critical
angle of total reflection at a binding layer using shadow line
analysis. Consequently, a non-metallic, optically transparent
element is used to immobilized the binding layer, thereby
simplifying immobilization procedures considerably. Moreover,
relatively low-cost instrumentation may be substituted for much
higher cost SPR biosensing devices. Accordingly, the present
invention saves both technician time and equipment expense.
[0126] It is contemplated to use the present invention for sensing
and monitoring a variety of binding interactions, including but not
limited to antigen/antibody, drug/receptor, polynucleotide
strand/complementary polynucleotide strand, aviden/bioten,
immunoglobulin/protein A, enzyme/substrate, and specific
carbohydrate/lectins interactions. Measurement output may be in the
form of a GO/NO GO report, for example by LCD display 38, as may be
useful in testing for the presence of E. coli. or other food born
pathogens. The present invention could also provide diagnostic
information, which is currently obtained by enzyme linked
immunosorbant assay (ELISA) and radio-immuno assays. The method and
apparatus of the present invention can be implemented in devices of
various sizes, ranging from hand held sensors to larger industrial
sensor systems. Applications of the method and apparatus of the
present invention also include sensing and monitoring of
environmental pollutants, pesticides, and metabolites, water
quality control, drug discovery, research and manufacture,
diagnosing chemical substance abuse, food and beverage
processing.
[0127] Although specific embodiments have been illustrated and
described herein, it will be appreciated by those skilled in the
art that any arrangement which is calculated to achieve the same
purpose may be substituted for the specific embodiment shown. This
application is intended to cover any adaptations or variations of
the present invention. Therefore, it is manifestly intended that
this invention be limited only by the following claims:
* * * * *