U.S. patent application number 09/882396 was filed with the patent office on 2001-10-11 for sensing system for specific substance and molecule detection.
This patent application is currently assigned to Accelr8 Technology Corporation. Invention is credited to Hanlin, H. John, Nunnery, David W., Robinson, MaryBeth, Starzl, Timothy W..
Application Number | 20010029050 09/882396 |
Document ID | / |
Family ID | 25230579 |
Filed Date | 2001-10-11 |
United States Patent
Application |
20010029050 |
Kind Code |
A1 |
Starzl, Timothy W. ; et
al. |
October 11, 2001 |
Sensing system for specific substance and molecule detection
Abstract
A system for detecting a specific substance or analyte of
interest is provided that includes one or more sensing units and an
instrument for analyzing the sensing units. Each sensing unit
preferably includes a substrate, an attachment layer and at least
one capture layer that includes a ligand layer. In one embodiment,
the attachment layer is tripartite and includes a lower binding
surface held to the substrate and an upper binding surface that
holds the ligand layer, together with an insulating layer disposed
between these two surfaces. The lower binding surface provides a
durable and stable attachment to the substrate. The upper binding
surface holds the ligand layer and does not jeopardize the
integrity or viability thereof. The insulating layer prevents any
unwanted interaction between the lower and upper binding surfaces.
Each sensing unit is supported on a test piece received by the
instrument. The instrument controllably positions the test piece
using marks and/or codes on the test piece. The instrument measures
a difference in mass in the sensing unit in connection with
determining whether or not the analyte of interest is present. A
light beam is used in making this measurement. In one embodiment,
multiple reflections of the light beam on the same sensing unit are
utilized to improve the sensitivity of the instrument. Mass
enhancement techniques are also preferably utilized to effectively
amplify the detected signal that is indicative of the analyte's
presence. The system can also include a device for heating,
humidifying and mixing materials of the sensing units, as well as
preventing cross-contamination of the sensing units.
Inventors: |
Starzl, Timothy W.;
(Boulder, CO) ; Nunnery, David W.; (Westminster,
CO) ; Robinson, MaryBeth; (Boulder, CO) ;
Hanlin, H. John; (Louisville, CO) |
Correspondence
Address: |
David F. Zinger
SHERIDAN ROSS P.C.
Suite 1200
1560 Broadway
Denver
CO
80202-5141
US
|
Assignee: |
Accelr8 Technology
Corporation
|
Family ID: |
25230579 |
Appl. No.: |
09/882396 |
Filed: |
June 14, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09882396 |
Jun 14, 2001 |
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09346039 |
Jul 6, 1999 |
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6274384 |
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09346039 |
Jul 6, 1999 |
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08820365 |
Mar 12, 1997 |
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5958704 |
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Current U.S.
Class: |
436/518 |
Current CPC
Class: |
Y10T 436/116664
20150115; G01N 33/525 20130101; Y10S 436/805 20130101; Y10S 435/808
20130101; Y10T 436/113332 20150115 |
Class at
Publication: |
436/518 |
International
Class: |
G01N 033/543 |
Claims
What is claimed is:
1. A sensing unit involved in detecting an analyte of interest,
comprising: a substrate having an outer surface; an attachment
layer for adhering to said substrate, said attachment layer used
for controlled binding of at least a first material and in which
said substrate outer surface has properties that cause unwanted
effects on the first material, said attachment layer including: a
lower binding surface having a material composition that adheres to
said substrate in order to provide a durable and stable lamination
of said attachment layer to said substrate; an upper binding
surface having a material composition different from said material
composition of said lower binding surface, said upper binding
surface including binding properties associated with said first
material which requires a controlled immobilization environment to
retain its functional characteristics; and an insulating layer
disposed between said lower binding surface and said upper binding
surface, said insulating layer having a material composition
different from each of said material compositions of said lower
binding surface and said upper binding surface and in which said
insulating layer acts to prevent the transfer of unwanted effects
from said lower binding surface to said upper binding surface and
from said upper binding surface to said lower binding surface;
wherein each of said material compositions of said lower binding
surface and said upper binding surface has properties that are
enhanced by the use of said insulating layer.
2. A sensing unit, as claimed in claim 1, further including: at
least one capture layer joined to said attachment layer for
providing an environment in which the analyte of interest, when
present, is properly immobilized to permit its subsequent
detection, said capture layer including said first material.
3. A sensing unit, as claimed in claim 1, wherein: each of said
material compositions of said lower binding surface and said upper
binding prevent it from being used for said material composition of
said insulating layer.
4. A sensing unit, as claimed in claim 1, wherein: said substrate
is other than a free electron metal.
5. A sensing unit, as claimed in claim 1, wherein: one of said
lower binding surface and said upper binding surface consists
essentially of inorganic monovalent compounds having linear
backbone structures.
6. A sensing unit, as claimed in claim 1, wherein: one of said
lower binding surface and said upper binding surface consists
essentially of at least one of organic monovalent and polyvalent
compounds and in which said compounds have particles that retain
their particle size of at least 40 microns or do not have said
particles.
7. A sensing unit, as claimed in claim 1, wherein: said lower
binding surface consists essentially of an organofunctional silial
compound.
8. A sensing unit, as claimed in claim 1, wherein: said attachment
layer includes some of the following materials:
6-azidosulfonylhexytriethoxy silane, aminoethyl-aminopropyl
trimethoxy silane, aminopropyltrioxysilane- ,
3-isocyanatopropyltriethoxysilane, phenyltriethoxysilane,
poly(ethylene)glycol, poly(ethylene)oxide, nitrocellulose,
paralene, nylon, polyester, polyamides, polyurethane, polystyrene,
avidin (and its derivatives), and biotin.
9. A sensing unit, as claimed in claim 1, wherein: said insulating
layer consists essentially of one or more materials from the
following group: polystyrene, polyurethane, and polyethylene
glycol.
10. A sensing unit, as claimed in claim 7, wherein: said
organofunctional silial compound includes amino-silane,
azido-silane and silanes comparable thereto.
11. A sensing unit, as claimed in claim 1, wherein: said substrate
consists essentially of a crystalline silicon.
12. A sensing unit, as claimed in claim 1, wherein: said first
material i s a specific material.
13. A sensing unit, as claimed in claim 1, further including: mass
enhancement means for amplifying a signal for detection related to
the presence of the analyte of interest.
14. A sensing unit, as claimed in claim 12, wherein: said mass
enhancement means includes one or more enzymes associated with such
attachment layer.
15. A sensing unit involved in detecting an analyte of interest,
comprising: a substrate other than a free electron metal and having
an outer surface; an attachment layer for adhering to said
substrate, said attachment layer used for controlled binding of at
least a first specific material and in which said substrate outer
surface has properties that cause unwanted effects on the first
specific material, said attachment layer including: a lower element
having a material that adheres to said substrate in order to
provide a durable and stable lamination of said attachment layer to
said substrate, said lower element consisting essentially of an
organofunctional silial compound; and an upper element having a
material composition different from said material composition of
said lower element, said upper element including an outer surface
including binding properties associated with said first specific
material which requires a controlled immobilization environment to
retain its functional characteristics; at least one capture layer
joined to said attachment layer for providing an environment in
which the analyte of interest, when present, is properly
immobilized to permit its subsequent detection, said capture layer
including said first specific material; wherein said material
composition of said upper element has properties that prevent it
from being used for said material composition of said lower
element.
16. A biosensor device, as claimed in claim 15, wherein: said
organofunctional silial compound includes amino-silane,
azido-silane and silanes comparable thereto.
17. A sensing unit, as claimed in claim 15, wherein: said upper
element consists essentially of one or more of the following:
polystyrene, polyurethane and polyethylene glycol.
18. A sensing unit, as claimed in claim 15, further including: mass
enhancement means associated with said attachment layer for
amplifying detection of the analyte of interest.
19. A sensing unit, as claimed in claim 18, wherein: said mass
enhancement means includes one or more enzymes provided with said
upper element.
20. A sensing system for obtaining information related to sensing
units, comprising: a test piece having a number of sensing units
and indicating means associated with each of the sensing units; a
housing having a number of walls and a receiver slot for receiving
said test piece wherein said test piece can move along a path of
said housing; a light beam assembly for obtaining information from
each sensing unit for use in determining the presence of a specific
substance of interest; and controller means located within said
housing for causing said test piece to move relative to said
housing path.
21. A system, as claimed in claim 20, wherein: said indicating
means includes indicia and said controller means includes a reader
assembly for reading said indicia in connection with stopping
movement of said test piece along said housing path.
22. A system, as claimed in claim 21, wherein: said indicia
includes an identification code that identifies at least one of the
sensing units.
23. A system, as claimed in claim 20, wherein: said controller
means comprises a test piece control assembly that includes a motor
disposed within said walls of said housing.
24. A system, as claimed in claim 20, further including: an input
unit provided on one of said walls of said housing for inputting
information related to the sensing units.
25. A system, as claimed in claim 20, further including: display
means located on one of said walls of said housing for displaying
information associated with the sensing units.
26. A system, as claimed in claim 20, further including: data
memory for storing data related to each of the sensing units
including identification data, time-related data and data related
to whether or not the analyte of interest is present.
27. A system, as claimed in claim 20, further including: at least a
first port communicating with an external computer system by which
said external computer system can download software for execution
by a processor of said controller means.
28. A system, as claimed in claim 20, wherein: said controller
means includes a processor for determining values related to the
sensing units and whether or not an analyte of interest is present
for each of the sensing units.
29. A system, as claimed in claim 20, wherein: said indicating
means includes at least one of a mark to indicate a location of
each of the sensing units and a code for identifying at least one
of the sensing units.
30. A system, as claimed in claim 20, wherein: said controller
means includes a number of electronic components including a
processor and in which said electronic components are held on a
printed circuit board and in which said printed circuit board is
located between said light beam assembly and an input unit for
providing input information used by said processor.
31. A system, as claimed in claim 20, wherein: said light beam
assembly includes polarizer means for receiving polarized light
having only one component when each sensing unit is tested to
determine whether the sensing unit has the analyte of interest and
in which said polarizer means remains stationary when said
polarized light is being received.
32. A system, as claimed in claim 20, wherein: said light beam
assembly generates a number of reflected light beams having
intensities for each of the sensing units, with each of said
reflected light beams being produced at an angle different from
perpendicular to each of the sensing units.
33. A system, as claimed in claim 32, wherein: said light beam
assembly includes a curved mirror that reflects and transmits light
and wave retarder means for providing additive polarization of said
reflected light intensities for each of the sensing units to
enhance sensitivity for determining the presence of the analyte of
interest.
34. A method for obtaining assay data, comprising: providing a test
piece having a number of sensing units; controlling movement of
said test piece; checking for identifying means on said test piece;
discontinuing movement of said test piece based on said checking
step; obtaining data related to a first sensing unit on said test
piece including determining whether an analyte of interest is
present using an optical detecting apparatus; and storing
information related to said determining step including
identification data related to the first sensing unit, time-related
data, and a determined value related to whether the analyte of
interest is present with the first sensing unit.
35. A method, as claimed in claim 34, wherein: said controlling
step includes regulating a motor using a processor that receives a
signal based on said identifying means.
36. A method, as claimed in claim 34, wherein: said identifying
means includes at least one of a first code that identifies a first
sensing unit and alignment indicia that indicates a position of at
least one of the sensing units.
37. A method, as claimed in claim 34, wherein: said providing step
includes inserting said test piece into a receiver slot of a
housing that contains said optical detecting apparatus and, before
said inserting step, locating said indicating means on said test
piece.
38. A method, as claimed in claim 34, wherein: said controlling
step includes regulating the turning on and turning off of a motor
using a processor and contacting said test piece using means for
engaging driven by said motor in order to move said test piece.
39. A method, as claimed in claim 34, further including: inputting
identification information related to at least one of the sensing
units using an input unit provided on one of the walls of a housing
that contains said optical detecting apparatus.
40. A method, as claimed in claim 34, wherein: said obtaining step
includes obtaining data for all of the sensing units on said test
piece and maintaining an angular position of polarizer means of
said optical detecting apparatus during receiving of light
intensities for all of the sensing units on said test piece, with
said light intensities being used to provide said data for all of
the sensing units.
41. A method, as claimed in claim 34, wherein: said obtaining step
includes collecting at least portions of light related to a number
of reflected light beams having intensities that are reflected from
areas of the first sensing unit and in which each incident light
beam from which each of said reflected light beams are generated is
at an angle other than perpendicular to the first sensing unit.
42. A device for heating and providing humidity to a number of
sensing units contained on a test piece, comprising: a heating
assembly for heating sensing units when a test piece containing
them is provided with said device; a humidifier assembly
communicating moisture to said heating assembly for humidifying the
sensing units; a barrier manifold for substantially preventing
cross-contamination of the sensing units when the test piece
containing them is provided with said device; and a mixing assembly
for mixing of materials in the sensing units when the test piece
containing them is provided with said device.
43. A device, as claimed in claim 42, wherein: said heating
assembly includes a plate sub-assembly that is heated for providing
heat by conduction to the test piece when it is provided with said
device.
44. A device, as claimed in claim 42, wherein: said heating
assembly includes a support frame that is joined to said plate
sub-assembly with both of said support frame and said plate
sub-assembly being pivotal.
45. A device, as claimed in claim 42, wherein: said humidifying
assembly includes water holding means located below said heating
means.
46. A device, as claimed in claim 45, wherein: said water holding
means includes at least a first sponge member positioned to a side
of said heating assembly and at least one well for containing water
adjacent to said first sponge member.
47. A device, as claimed in claim 42, wherein: said barrier
manifold includes a number of spaced barrier members that act to
separate each of the sensing units from the other sensing units
when the test piece containing the sensing units is positioned in
said device.
48. A device, as claimed in claim 42, wherein: said barrier
manifold is pivotal and applies a pressure to the test piece when
the test piece is positioned in said device.
49. A device, as claimed in claim 42, wherein: said mixing assembly
includes a motor that is positioned at an angle to enhance the
mixing of the materials in the sensing units.
50. A device, as claimed in claim 49, wherein: said angle is
greater than about 10 degrees but less than about 70 degrees.
51. An instrument for detecting an analyte of interest, comprising:
first means for supporting a sensing unit to be tested; second
means for locating the sensing unit in a desired position; and
third means for determining whether the analyte of interest is
present with the sensing unit being tested, said third means
including an optical detecting apparatus for generating a number of
light reflections from the sensing unit, with each of said light
reflections being at an angle other than 0.degree., 90.degree. and
Brewster's angle relative to said first means, said third means
including collection means for collecting at least portions of said
light reflections and using said collected light reflections in
determining whether the analyte of interest is present.
52. An instrument, as claimed in claim 51, wherein: said means for
generating includes a mirror and a wave retarder plate, said wave
retarder plate being disposed in a path of said light reflections
between the sensing unit and said mirror and wherein said wave
retarder plate causes said light reflections that pass through it
to be additive of polarization change.
53. An instrument, as claimed in claim 52, wherein: said mirror
includes curved portions and in which said mirror has optical power
and a reflective coating, with portions of said light reflections
received by said mirror being transmitted through said mirror.
54. An instrument, as claimed in claim 52, wherein: said mirror has
a reflective coating and said wave retarder plate is a portion of
said reflective coating.
55. An instrument for detecting an analyte of interest, comprising:
first means for supporting a sensing unit to be tested; second
means for locating the sensing unit in a desired position; and
third means for determining whether the analyte of interest is
present with the sensing unit, said third means including an
optical detecting apparatus that has means for generating polarized
light having only one component and means for extinguishing said
one component, wherein said means for generating and said means for
extinguishing remain fixed in position during the time that said
optical detecting apparatus provides light to the sensing unit.
56. An instrument, as claimed in claim 55, wherein: said one
component includes one of s-polarization and p-polarization.
57. An instrument, as claimed in claim 55, wherein: said means for
generating includes a linear polarizer that receives light prior to
said light being incident upon the sensing unit and said means for
extinguishing includes a detector polarizer that receives light
reflected from the sensing unit.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to detection and/or
quantitation of an analyte of interest or specific substance and,
in particular, to a system that includes an instrument for
measuring mass associated with a multi-layered sensing unit to
determine whether an analyte of interest is present.
BACKGROUND OF THE INVENTION
[0002] Sensors and methods for the detection of the presence of
substances or molecules in a sample, using a solid-phase assay
system, have been described previously. Typically, the sample is
put in contact with the sensor, allowing analyte present in the
sample to bind to the analyte-specific ligand layer of the sensor.
For analysis, the sample may be removed from the sensor. The sensor
surface is then analyzed for the presence of the analyte. Sensors
can be defined as including immunosensors, affinity sensors and
ligand binding sensors, each of which is characterized as involving
specific mass change activity in connection with determining
whether or not certain molecules or substances are present. Sensors
are typically efficient at binding the substance of interest
(analyte) and highly sensitive and specific to the analyte. The
sensor may consist of one or several layers of various chemical and
physical compositions. The composition depends on the nature of the
analyte and the matrix in which the analyte is contained. These
layers may include any combination of: a solid supporting
substrate; attachment layer or layers to bind the substrate and/or
subsequent layers in the sensors any number of intermediate layers;
a ligand layer that binds specifically to the analyte. Detection of
the analyte bound to the sensor can be achieved by several means
including, but not limited to, electrochemical, chemical, and
optical methods. Detection of the analyte may be enhanced by
various means including enzyme amplification, and the use of a
mass-enhanced analyte-specific secondary ligand.
[0003] A solid substrate, or base, of the sensor has inherent
physical, chemical, electrical, or optical properties that are
suited specifically to the detection method employed in the assay.
A ligand layer is typically provided above the substrate and an
analyte to be detected or measured is bound to the ligand layer.
The solid support may be used for the direct binding of the analyte
to its surface and subsequent detection. However, depending on the
composition, complexity, and/or stability of the analyte or the
sample in which the analyte is contained, and the nature of the
interactions of the sample/analyte with the solid substrate, it may
be necessary to add one or several intermediate layers to the solid
support.
[0004] Attachment layers may be used as intermediate layers between
the solid substrate and the ligand layer if, for example, the
ligand layer does not adhere to the substrate, or is destroyed,
denatured, destabilized or otherwise inactivated upon binding to
the substrate. The surface of the intermediate layer in contact
with the solid substrate must adhere tightly to the substrate
throughout the preparation and use of the test piece. The surface
of the intermediate layer opposite the solid substrate must either
be suitable for strong binding of either the ligand layer or
another intermediate layer. In this manner, and using these
considerations for the nature of the intermediate layers, multiple
layers may be assembled, the topmost of which is suitable for the
binding of the ligand layer.
[0005] The ligand layer forms a sensing surface that is receptive
specifically to the analyte of interest when the analyte is present
in a sample to be tested. The analyte is thus immobilized onto the
sensing surface of the sensor and can be detected by any of the
methods mentioned above.
[0006] Although some multi-layered sensors such as those outlined
above have been described previously in the prior art, development
of such sensors, in accordance with the present invention, seeks to
improve and enhance their sensitivity. The goal of one element of
the present invention, in order to improve this sensitivity, is to
immobilize a ligand layer that retains maximum binding capacity for
a specific analyte. This usually involves the use of an
intermediate attachment layer or layers as described above. This
multi-layer molecular film is designed specifically to accommodate
downward interaction with the substrate, and upward, optimized
interaction with the ligand layer.
[0007] Detachment, or delamination, of these intermediate layers
from the supporting substrate is a serious problem that must be
solved to successfully build a multi-layer sensing surface.
Delamination occurs between the substrate and an intermediate layer
if the composition of these two components is not conducive to a
strong physical or chemical interaction between the components. The
interaction between the substrate and intermediate layer may be
weakened during the manufacture, assembly, transport, preparation
or use of the sensor.
[0008] Once an attachment layer or layers is produced that is
stable to delamination from the solid support, the topmost of the
intermediate layers is used to immobilize a ligand layer specific
to the analyte of interest. It is critical that the topmost
attachment layer optimizes its interaction with the ligand layer in
order to provide maximum binding capacity for the analyte of
interest and prevent denaturation, deactivation, or inactivation of
the ligand layer. These provisions for the immobilization of the
ligand layer are essential to the enhanced sensitivity of the
test.
[0009] Known sensing systems, in addition to multi-layered sensors
for immobilizing analyte that may be present in a test sample,
include instrumentation for detecting the immobilized analyte. One
class of instrumentation, including surface acoustic wave
spectroscopy, ellipsometry, and quartz microbalances, measures the
change in mass of the sensor upon immobilization of an analyte.
Generally speaking, when the analyte is present in the test sample,
it can be detected based on a change in mass at the surface as
compared to the mass when no analyte is present in the sample.
[0010] Optical instruments in this class, as described in the prior
art, direct a beam of light through a number of instrument
components or elements to the sensing surface that has been
previously exposed to the sample being tested. Light is reflected
from the sensor, and its reflected properties, including intensity
and various optical properties may be measured. Any change in mass
of the sensor due to analyte binding is represented by a change in
the properties of the reflected light. In particular, measuring
changes in polarization state of the reflected light has proven to
be a highly sensitive measurement of mass change. Briefly, the
sensor may be analyzed by an appropriate instrument that detects
and/or measures the presence of the analyte using light reflected
from the analyte or light that is transmitted through the
analyte.
[0011] The main problem associated with instruments that employ
these optical techniques for detection of surface bound analyte
involves the accuracy of detection. Since extremely small changes
in mass may be indicative of the presence of the analyte, it is a
goal of the present invention that the instrument be highly
sensitive to enable it to detect such mass changes. Additionally,
because these instruments are expected to be utilized in a variety
of environments outside of well-controlled laboratory settings, it
is a further goal that the instrument design take into
consideration a number of factors such as component size,
durability and automation of instrument operation.
[0012] Based on the foregoing factors and considerations, it would
be advantageous to devise a sensor system that overcomes such
drawbacks or deficiencies of the prior art by providing a system
that includes an instrument that readily functions and cooperates
with a sensing unit for detection and/or measurement of specific
mass change activity due to the presence of an analyte of interest.
The instrument of such a system would be highly sensitive and
accurate in connection with the detection and/or measurement
related to mass change, while the sensing unit of such a system
would immobilize a ligand layer that retains, for all necessary
purposes, the analyte of interest.
SUMMARY OF THE INVENTION
[0013] In accordance with the present invention, a sensing system
is provided that includes a sensing unit and associated processes
for making, assembling, and using such a sensing unit. The sensing
system also includes a test piece on which is positioned one or
more sensing units capable of capturing and immobilizing an analyte
or analytes of interest from a test sample, and an instrument for
detecting the analyte(s) immobilized on the sensing unit. The test
sample containing the analyte of interest may take the form of a
true vapor, a liquid or an extracted solid.
[0014] In one embodiment, the sensing unit includes a solid
reflecting substrate, an analyte-specific ligand layer and a
tripartite attachment layer used to immobilize the ligand layer.
The solid substrate is preferably any reflective substance other
than a free electron metal that provides a base of the sensing
unit. The upper surface of the solid substrate would, if improperly
isolated from the ligand layer, cause denaturation, decay, or
inactivation of the ligand layer. The tripartite attachment layer
is defined by a lower binding surface, an insulating intermediate
layer, and an upper binding surface.
[0015] The tripartite attachment layer used to immobilize the
ligand layer may consist of three distinct materials, or different
material compositions of a single material, providing that each
element of this layer (the lower binding surface, the upper binding
surface, and the insulating layer) has properties that are suited
particularly for its role in the tripartite attachment layer.
[0016] The lower binding surface contacts the surface of the solid
substrate and is comprised of a material that adheres tightly to
the solid substrate. This lower binding surface provides a durable
and stable lamination of the entire attachment layer to the silicon
surface. The upper binding surface of the tripartite attachment
layer has chemical or physical properties that allow it to
immobilize an analyte-specific ligand layer. The insulating layer
is located between the lower and upper binding surfaces.
Significantly, the insulating layer acts to prevent the transfer of
various effects from the lower binding surface to the upper binding
surface and, additionally, from the upper binding surface to the
lower binding surface. The insulating intermediate layer protects
the ligand layer from the various effects of the solid substrate.
The analyte-specific ligand layer is in contact with the upper
binding surface of the tripartite attachment layer. This ligand
layer captures and immobilizes a specific analyte when it is
present in a sample to be tested. The sensing unit preferably has a
number of associated attributes including that each of the material
compositions of the lower binding surface and the upper binding
surface has properties that are preserved with the use of an
effective insulating layer. The tripartite attachment layer may
consist of three distinct materials, or different material
compositions of a single material, providing that each element of
this layer (the lower binding surface, the upper binding surface,
and the insulating layer) has properties that are suited
particularly for its role in the tripartite attachment layer.
[0017] In a related variation, the upper surface of the tripartite
attachment layer may be used to bind to a ligand layer that is
receptive to or captures a non-specific analyte. That is, the
tripartite attachment layer is not limited to use with an
analyte-specific ligand layer.
[0018] In another embodiment, the sensing unit includes the solid
substrate of the first embodiment, such as a reflective substrate
other than a free electron metal (e.g. crystalline silicon), two
elements or layers of an attachment layer, and an analyte-specific
ligand layer. The attachment layer element closest to the silicon
surface is comprised of, for example, an organofunctional silial
compound. The silane portion of the attachment layer forms a
covalent linkage to the silicon substrate surface, leaving the
reactive organofunctional group available for further interaction
with a second element of the attachment layer. The second element
of the attachment layer is usually an organic polymer film that is
applied, preferably spin coated, to the organofunctional silial
compound layer. This organic polymer film provides a controlled
environment to immobilize the analyte-specific ligand layer or to
attach the non-specific sample to serve as an attachment platform
for the non-specific binding of analyte in the sample.
[0019] A sensing surface may include the solid silicon substrate of
the first embodiment, a single attachment layer applied using
conditions that prevent the attachment layer from delaminating from
the solid substrate, and an analyte-specific ligand layer. The
attachment layer is, for example, an organic polymer. This single
attachment layer provides all three functions described in the
first embodiment: binding to the silicon support, immobilizing the
ligand layer, and isolating the ligand layer from properties of the
solid substrate that may inactivate the analyte-specific ligand
layer. The ligand layer captures and immobilizes a specific analyte
when it is present in a test sample.
[0020] Like the tripartite attachment layer, both the dual element
attachment layer and the single attachment layer may be used to
bind to a ligand layer that is receptive to a non-specific
analyte.
[0021] Detection of the analyte may be improved further through
various means of mass enhancement. These mass enhancement
techniques typically involve the secondary binding of an
analyte-specific ligand that may or may not be different from the
ligand used to immobilize the analyte on the surface. This
secondary binding ligand may be further linked to an additional
amplification system. These additional amplification systems
include, but are not limited to, enzymes (such as horse radish
peroxidase or alkaline phosphatase), macromolecules (such as DNA,
RNA or ferritin) or small particulates (such as polystyrene
microspheres, metal sols, silica, self-assembling monolayers or
film-forming compounds).
[0022] Each sensing unit is adapted to be held at a defined
location on a test piece or slide. Typically, each test piece is
able to support a predetermined number of sensing units. The
defined positions on the test piece include marks, bar codes, or
other indicia that are useful in identifying the particular sensing
unit on a test piece. The test piece is of a size and shape to be
used with and received by an instrument for detecting and/or
measuring, when present, an analyte or substance of interest that
is bound to one or more of the sensing units on the test piece. The
instrument includes a compact housing that includes a number of
assemblies or elements. A test piece movement assembly may provide
automatic control of the positioning of the test piece,
particularly relative to a light beam assembly that is used in
detecting whether or not an analyte of interest is present on the
current sensing unit being tested.
[0023] The instrument may also include a reader assembly for
reading the marks or codes on the test piece and regulating the
movement of the test piece using the application of power to a
motor in order to properly position the test piece and,
concomitantly, the sensing unit under test, relative to the light
beam assembly.
[0024] The housing includes an aperture for receiving the test
piece. The housing may further include a user input assembly, such
as a keypad, for requesting information and data related to the
detection process. The instrument also has a display unit, for
example LCD, that displays requested output information, such as
the results of the detection process, as well as graphs or plots.
The display unit is also useful in communicating available options
to the technician or user related to information and data that is
available from the instrument. Preferably also, the instrument is
able to communicate with an external processing source or computer
system that enables the instrument to download data or other
information related to the functions that it performs in connection
with the detection and/or measurement process.
[0025] The light beam assembly includes a source of light that is
controlled and directed for obtaining information useful in
analyzing the mass of the sensing unit in connection with
determining whether or not the analyte of interest is present. In
one embodiment, the light beam assembly includes components for
providing a number of bounces or reflections of the altered light
through the same sensing unit in order to amplify the changes in
the reflected light to improve sensitivity. Such sensitivity is
particularly advantageous when the substance of interest is present
and the mass change of the sensing unit is relatively small and
difficult to detect.
[0026] With respect to further aspects related to the method or
operation for determining whether or not an analyte of interest is
present, the test piece having a number of sensing units is
inserted into the instrument housing. The test piece movement
assembly is controlled by a digital controller having a processor
so that it is moved inwardly within the housing until the reader
assembly reads a mark or code that is interpreted by the processor,
and thus, controls the powering of f of the motor of the test piece
movement assembly. The light beam assembly provides a beam of light
that is controlled and caused to reflect from the sensing unit
being tested. Reflected light from this particular sensing unit is
collected and analyzed. In certain embodiments for obtaining light
from a sensing unit, the instrument is also configurable to provide
virtually only s-polarized light or, alternatively, p-polarized
light. In such cases changes in the detected signal due to change
in polarization state from a particular sensing unit will be a
function of any increase or decrease in the mass of the sensing
unit. Preferably, such light is linearly polarized; however,
circular or elliptical states of polarization can also be utilized.
The results of the analysis are stored and available for
presentation and/or for the user to obtain and make decisions based
on such results. The stored information includes not only
information related to the result of whether or not the analyte of
interest is present, but also information related to the
identification of the individual test pieces and sensing units.
[0027] In one embodiment of the invention, the system further
includes a device that has a number of assemblies for providing
desired functions associated with test pieces or test performance.
A heating assembly includes a number of plates that are heated to
enable heat to be conducted to the test piece. The heating assembly
also includes a support frame that permits it and the heated plates
to be pivoted for access to a number of absorbative members of a
humidifying assembly. Each of the absorbative members is soaked
with water to enable moisture or vapor to be developed due to the
heat generated by the heating assembly. A mixing assembly is also
part of the device for mixing the materials associated with the
sensing units. A barrier manifold having a number of barrier
members is pivotally movable relative to the test piece when it is
positioned within the incubation device. The barrier members are
useful in avoiding cross-contamination among the number of sensing
units with the test piece.
[0028] Based on the foregoing summary, a number of key features of
the present invention are easily recognized. A system is provided
that includes a sensing or assay unit comprising an attachment
layer that both substantially reduces the possibility of
delamination of the attachment layer from a substrate and
immobilizes a ligand layer that is receptive to an analyte of
interest when it is present with the sensing unit being tested. In
one embodiment, the attachment layer is definable as three layers
that include an insulating layer for preserving the integrity and
proper functioning of the layers or surfaces that the insulating
layer contacts.
[0029] The system also may include methods and materials for
enhancing the detection and/or measurement of mass change activity
of the substance of interest, when present. Such mass enhancement
techniques are associated with the sensing unit and assist an
appropriate instrument in its detecting or measuring operations by
effectively enhancing the presence of the property (mass) being
measured.
[0030] The system further includes an instrument for receiving a
test piece having a number of sensing units in order to determine
whether or not one or more of them contains an analyte of interest.
The instrument is compact, easy to use in connection with obtaining
desired data or other information and has numerous automatic
operational features. In particular, the instrument is able to
control the position of the test piece to properly align it with a
beam of light to be used for detection purposes, conduct the
necessary test including analysis of the particular sensing unit
and store the results of the test including an identification of
the particular sensing unit and the time of the testing. In one
embodiment, light is caused to reflect a number of times on the
same sensing unit to increase the sensitivity of the instrument. In
other variations, only s-polarized light or p-polarized light is
obtained to enhance the sensitivity of the detected signal from a
sensing unit. The instrument is also able to store and download
data to an external computer terminal for additional evaluation or
use of the obtained data. Preferably, the system also includes a
device for housing the test piece which is beneficial in
controlling temperature, humidity and mixing the materials provided
with or as part of the sensing unit. This device includes a barrier
manifold that acts to prevent each of the tests samples from
contaminating each of the other samples.
[0031] Lastly, an overall sensing system is provided that has all
of the necessary sub-system components for detecting and/or
measuring a substance of interest by relying on mass change
activity. These sub-system components readily cooperate and
function together in meeting such main objectives. Each sensing
unit is properly formed and prepared for testing, including use of
suitable mass enhancement techniques that effectively amplify the
signal that is detected when the analyte is present. The sensing
units on the test piece are properly prepared and accurately
positioned relative to the instrument for conducting the desired
test. The instrument itself is highly sensitive to mass activity
change in connection with determining whether or not the analyte of
interest is present.
[0032] Additional advantages of the present invention will become
readily apparent from the following discussion, particularly when
taken into account with the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIG. 1 is a diagrammatic representation of a sensing unit
that includes a tripartite attachment layer;
[0034] FIG. 2 is a diagrammatic representation of the sensing unit
of FIG. 1 illustrating the resultant sensing unit after certain
process steps have been conducted and when the analyte of interest
is present;
[0035] FIG. 3 is a diagrammatic representation of the sensing unit
of FIGS. 1 and 2 in which a mass enhancement composition is also
utilized;
[0036] FIG. 4 diagrammatically illustrates a particular sensing
unit that has a dual attachment layer;
[0037] FIG. 5 diagrammatically illustrates the sensing unit of FIG.
4 after certain process steps have been conducted and in which the
analyte of interest is present;
[0038] FIG. 6 diagrammatically illustrates the sensing unit of
FIGS. 4 and 5 in which a mass enhancement composition is also
utilized;
[0039] FIGS. 7 and 8 are flow diagrams related to the making and
use of the sensing unit of FIGS. 4-6;
[0040] FIG. 9 illustrates an exploded view of an instrument of the
present invention involved in the detection and/or measurement
process;
[0041] FIG. 10 is a block diagram of major components of the
instrument used in analyzing the sensing units and in controlling
positioning thereof;
[0042] FIG. 11 is a perspective view of a test piece;
[0043] FIG. 12 is an enlarged, diagrammatic view of the light beam
assembly, the test piece movement assembly and the reader assembly
used in an instrument of the present invention;
[0044] FIG. 13 is an exploded view illustrating a number of
components of the light beam assembly, the test piece movement
assembly and the reader assembly of the embodiment of FIG. 12;
[0045] FIG. 14 is a flow diagram setting out major steps related to
the operation of the instrument using one embodiment of a light
beam assembly;
[0046] FIGS. 15A-15B are flow diagrams setting out steps related to
conducting analyses of a number of sensing units using two test
pieces;
[0047] FIG. 16 diagrammatically illustrates a test piece having a
number of sensing units in a desired position under control of the
instrument of the present invention;
[0048] FIG. 17 illustrates another embodiment of an instrument that
includes a test piece control assembly of a second embodiment;
[0049] FIG. 18 diagrammatically illustrates another view of the
embodiment of FIG. 17 in which further details of the test piece
control assembly are shown;
[0050] FIG. 19 is a side elevational view of the embodiment of
FIGS. 17 and 18;
[0051] FIG. 20 illustrates a housing for enclosing the embodiment
of FIGS. 17-20;
[0052] FIG. 21 is a diagrammatic view of another embodiment of a
light detection assembly that employs a multi-bounce operation on
the same sensing unit in connection with amplifying the reflected
light associated with determining whether an analyte of interest is
present;
[0053] FIG. 22 is a flow diagram setting out major steps related to
the operation of the instrument using the embodiment of FIG.
21;
[0054] FIG. 23 illustrates a perspective view of a device for
containing test pieces that performs a number of functions; and
[0055] FIG. 24 is an exploded view of the device of FIG. 123.
DETAILED DESCRIPTION
[0056] With reference to FIG. 1, an embodiment of a sensing or
assay unit 100 of the system of the present invention is
schematically illustrated. As seen, the sensing unit 100 includes a
number of layers made of different materials. The multi-layered
unit 100 provides a number of functions as will be described later
herein. A sample 104 is deposited on the unit 100. The sample 104
is to be subsequently tested to determine whether or not it
contains a specific analyte or substance of interest. The specific
analyte to be tested to determine whether it is present can include
any one of a number of different substances, materials and/or
macromolecules, such as biological materials, chemicals, peptides
and toxins. In one area of applications, the sensing unit 100 is
used as part of a testing system and procedure to test for the
presence of unwanted or harmful substances in animal or human body
fluids. Depending on the results of such testing, appropriate
action or steps can be taken.
[0057] The layers of the sensing unit 100 include a substrate or
base layer 108 that is used as a support member and is made of a
material that is useful or compatible with the testing instrument
and protocol. In a preferred embodiment, the substrate 108 is other
than a free electron metal (if a substrate has characteristics of a
free electron metal, it is not acceptable and cannot be used), but
is preferably made of a crystalline silicon material that has
desired properties for use with a testing instrument that utilizes
reflected light in determining whether the analyte of interest is
present.
Tripartite Attachment Layer
[0058] The substrate 108 has an attachment layer 112 joined thereto
that is used to immobilize a ligand layer. The attachment layer 112
includes material compositions for performing two main functions.
The attachment layer 112 must securely attach or join to the
substrate 108 and it must immobilize, without harm or unwanted
alteration, one or more capture layers including the ligand layer
116. The ligand layer 116 comprises a known or specific material or
macromolecule that properly bonds with the analyte of interest when
it is present in the test sample. The ligand layer 116 may comprise
a number of different materials such as monoclonal and polyclonal
antibodies, antigens, avidin, biotin, nucleic acids, proteins,
peptides, and receptors. In a variation of this embodiment, instead
of using ligand layer 116, or any other capture layer, there is a
direct binding of the analyte, when present, to the tripartite
attachment layer 112.
[0059] In one embodiment, the attachment layer 112 is tripartite,
non-homogeneous and non-porous. Such an attachment layer 112
includes a lower binding surface 118, an upper binding surface 120
and an insulating or intermediate layer 124 disposed between the
surfaces 118, 120. Unlike known prior art, the tripartite
attachment layer 112 includes three layers having differing
material properties for performing a number of functions associated
with establishing desired bonding and insulating functions. The
lower binding surface 118 is characterized by a material
composition that causes it to be adequately bonded to the substrate
108. Such a bonding adequately resists detachment or delamination
of the lower binding surface 118 from the substrate 108. The lower
binding surface 118 actively adheres or displays enhanced binding
attributes to the substrate 108 through one or more binding
mechanisms or techniques, such as adsorption, electrostatic,
chemical, covalent or ionic means. Preferred techniques or means
provide a durable and stable lamination of the attachment layer 112
to the substrate 108. The upper binding surface 120 preferably
consists of a material or materials that are different from the
material composition of the lower binding surface 118. The upper
binding surface 120 requires different attributes or properties
since its primary function is to provide a proper binding
environment for the ligand layer 116 so that the ligand layer 116
is not subject to deformation, denaturation, stearic alteration, or
partial or total inactivation of the ligand(s) that make up the
ligand layer 116. Typically, such macromolecules require a
controlled immobilization environment to retain such functional
attributes. A failure to retain such attributes results in an
inability of the ligand layer 116 to bind to a viable analyte of
interest when it is present.
[0060] The insulating layer 124 is a protective or barrier layer
between the lower and upper binding surfaces 118, 120. This layer
124 has two key functions. The insulating layer 124 prevents
unwanted effects or activities to be transmitted in each of two
directions. In a first direction, unwanted or potentially harmful
effects on the upper binding surface 120 due to the material
composition of the lower binding surface 118 or the substrate 108
are controlled or prevented by the insulating layer 124. In a
second direction, such effects on the lower binding surface 118 or
the substrate 108 due to the material composition of the upper
binding surface 120 are prevented from occurring. The unwanted
effects involve the transfer of potentially harmful electrostatic,
ionic, hydrophobic or covalent properties to one of the two
surfaces 118, 120 from the other of the two surfaces. The
insulating layer 124 also prevents the transfer of unwanted effects
that may be attributable to the capture layer including ligand
layer 116 or any other layer, material or sample that may be
included with the sensing unit 100.
[0061] Each of the lower and upper binding surfaces 118, 120 has a
material or materials composition that is different from the
material or materials composition of the insulating layer 124. Each
of these two binding surfaces 118, 120, requires properties in
order to properly function that are different from the insulating
or protective layer. The insulating layer 124 may be homogeneous or
may be non-homogeneous, such as including a gradient of different
materials. Although the tripartite attachment layer 112 may be
non-porous, or effectively non-porous, and resists permitting
liquid to flow through it, the insulating layer 124 is the region
or section of the tripartite attachment layer 112 that inhibits
such passage of liquid. The lower and upper binding surfaces 118,
120 need not be impervious or non-porous. Related properties of the
insulating layer 124 include its ability to resist or oppose
delamination forces or activities for relatively long periods of
time.
[0062] The compositions of the lower binding surface 118, the upper
binding surface 120 and the insulating layer 124 can include one or
more of different materials or groups of materials that are
appropriate in performing the functions and including the
properties required of such surfaces and layer. In one embodiment,
the substrate 108 includes, for example, commercially available
crystalline silicon that may or may not have a native oxide layer.
The tripartite attachment layer 112 can include any combination of,
for example, the following materials: inorganic or organic
monomeric and polymeric compounds. Examples of materials that can
by utilized for the three sections of the tripartite attachment
layer include: 6-azidosulfonylhexyltriethoxy silane,
aminoethylamino-propyl trimethoxy silane,
aminopropyltriethoxysilane, 3-isocyanatopropyltriethoxysilane,
phenyltriethoxy-silane, poly(ethylene)glycol, poly(ethylene)oxide,
nitrocellulose, paralene, nylon, polyester, polyimides,
polyurethane, polystyrene, avidin (and its derivatives), and
biotin, and any combination thereof.
[0063] In certain embodiments, the attachment layer 112 has the
following: inorganic monovalent (non-polymeric) compounds and/or
organic monovalent and/or polyvalent compounds that either do not
have particles or do have particles that retain their particle size
(do not coalesce) of at least 40 microns and each of these has
linear backbone (non-branching, random and/or irregular) structures
including, for example, polystyrene, polyurethane, polyethylene
glycol, avidin-biotin, and/or combinations thereof.
[0064] With reference to FIG. 2, a further depiction of the sensing
unit 100 is provided that illustrates the state of the sensing unit
100 after a number of assay process steps have been conducted that
will be described in greater detail later herein in the context of
a second embodiment of a sensing unit but which steps are also
applicable to the embodiment of FIG. 2. Essentially the only
substance remaining after such steps is the analyte of interest
106, when present. The analyte of interest 106 is bound or
immobilized on the accepting or capturing ligand layer 116. At this
stage of its preparation, the sensing unit 100 can be positioned
for testing using an appropriate instrument. Alternatively and
preferably, as seen in FIG. 3, one or more mass enhancement
substances or materials 110 used effectively amplify the mass
change, when present, that is being detected or measured. These
mass enhancement techniques can take one or more different forms
including kinetic-active mass enhancement, passive mass enhancement
and a self-assembling amplification system. Mass enhancement
substances will be subsequently described in connection with
certain examples. These mass enhancement substances improve the
detection of the analyte of interest 106 when it is present and
usually involve the secondary binding of an analyte-specific
ligand, which may be further joined to additional amplification
systems.
Dual Element Attachment Layer
[0065] Another embodiment of a sensing unit is schematically
illustrated in FIG. 4. This embodiment includes a dual element or
laminate attachment layer, instead of a tripartite attachment
layer. This dual element layer, unlike known prior art, is made of
different materials including an organofunctional silial compound
and an upper element that attracts or bonds to a desired ligand.
This sensing unit 150, like the first embodiment, includes a
substrate 154 made of a silicon-based material such as a silicon
wafer. The attachment layer 158 includes a lower element 162 that
has an organofunctional silial compound including, for example,
6-azidosulfonylhexyltriethyoxy silane and
aminopropyl-triethoxysilane.
[0066] The lower element 162 has properties like the lower binding
surface 118 of the first embodiment including primarily providing a
durable and stable attachment to the substrate 154. The dual
attachment layer 158 also includes an upper element 166 that is
bound to the lower element 162 by one or more of an adsorption,
covalent, ionic, chemical and/or electrostatic attachment.
Importantly, the upper element 166 must have an outer surface 168
that causes a capture layer including a ligand layer 170 to be
bound thereto. In the dual attachment layer embodiment, the outer
surface 168 and the remaining portions of the upper element 166 are
homogeneous. The capture layer including ligand layer 170 has the
same functions and properties and can be comprised of the same
material compositions as the capture layer of the first embodiment.
A test sample 174 is provided on the ligand layer 170, similar to
that in which the test sample 104 is provided on the ligand layer
116 of FIG. 1.
[0067] Like the embodiment of FIGS. 1-3, the sensing unit 150 is
prepared for testing whether or not an analyte of interest is
present. As seen in FIG. 5, after certain process steps that will
be subsequently discussed, the analyte of interest 156 is
illustrated as being bound to the ligand layer 170. At this stage,
the sensing unit 150 may be positioned for testing using, for
example, the instrument and variants thereof that will be described
later herein. Preferably, mass enhancement systems or substances
160 are provided on the analyte of interest 156. Such a mass
enhancement system 160 performs the same functions that are
provided in the embodiment of FIGS. 1-3.
[0068] With regard to further details of the composition and method
of making the dual element embodiment, reference is made to FIGS. 7
and 8. As provided in the flow diagrams, together with subsequent
Examples, 1-3, a number of steps are involved in the making of the
sensing unit 150. In particular with reference to FIG. 7, at step
200, a crystalline silicon wafer is obtained. At step 202, the
wafer is positioned in a spin-processor for subsequent receipt of
the dual element attachment layer. Next, at step 206, an
organofunctional silial compound, such as
6-azidosulfonylhexyltriethoxysilane, is spin coated onto the wafer.
At step 208, an organic polymer, such as polystyrene, is next spin
coated onto each lower element 162 to form each upper element 166.
As described at step 212, the wafer and the lower and upper
elements 162, 166 are then baked at a sufficient temperature for a
suitable time period in order to cure them.
[0069] As noted at step 214, the wafer is cut into strips to form
each dual element attachment layer 158 that includes the lower and
upper elements 162, 166.
[0070] In accordance with step 216, a number of sensing units are
then mounted on a glass slide. At step 218, a masking step is
performed by which each sensing unit is separated from the others
in a manner that reduces potential contamination between or among
the sensing units, as well as such placement and separation of the
sensing units being compatible with the placement and location of
identifying bar codes or other indicia.
[0071] The dual element attachment layer 158 may be tested for
stability at step 220 but need not be. In particular, the
attachment layer 158 may be tested to determine its ability to
resist or oppose delamination forces or activities.
[0072] At step 222, a primary ligand layer 170 is coated to the
outer surface of each sensing unit 150. As set out at step 224, the
dual element attachment layer 158 and accompanying ligand layer 170
are then baked at a determined temperature for a sufficient time in
order to incubate the primary ligand layer 170. Next, at step 226,
any additional primary ligand binding sites in each sensing unit
are blocked to prevent unwanted, non-specific binding. Such
blocking is usually preceded by a rinsing and drying of each
sensing unit 150.
[0073] With reference to FIG. 8 at step 230, a test sample is then
deposited onto each sensing unit 150. The primary ligand layer 170
comprises a known or specific molecule that properly bonds with the
analyte of interest when it is present. After adding the test
sample, a further incubation is conducted at step 234 in order to
allow specific binding of the analyte, when present, to occur.
[0074] About the time the testing or assay is to be performed, the
blocking composition is rinsed away and, preferably, a secondary
ligand is added, in accordance with step 238. At step 242, each
sensing unit 150 is once again incubated including, in this
embodiment, the primary and secondary ligands and an analyte of
interest. As previously noted, it is typically beneficial to
include a mass enhancement composition. Hence, at step 246, after
the incubation and secondary ligand binding, a further rinsing is
conducted and then a mass enhancement composition is added, such as
an enzyme substrate. Now each sensing unit 150 is ready to be
utilized with a detecting or measuring instrument to read or detect
any change in mass in the particular sensing unit 150 due to the
analyte of interest, when present, at step 250.
[0075] Although the foregoing description relates to the method of
making the dual element attachment layer, such steps are equally
applicable to a process for making the tripartite attachment layer,
except where differences may arise due to the tripartite nature of
the tripartite attachment layer, as compared to the dual element
attachment layer.
[0076] Further detailed explanations of the steps denoted in FIGS.
7 and 8 are provided in the following Examples in which Example 1
relates substantially to the steps of FIG. 7 and Examples 2 and 3
relate to the steps of FIG. 8 and some of the steps of FIG. 7.
EXAMPLE 1
[0077] A batch of monocrystalline silicon wafers was obtained
having polished silicon wafer surfaces with surface native dioxide
thicknesses. The wafers were then positioned in a spin-processor
for receipt of the dual element attachment layer.
[0078] A 2% 6-azidosulfonylhexyl triethoxysilane (azidosilane) in
solution of 95% 200 proof ethanol and 5% dH.sub.2O with 1 drop 1N
sodium hydroxide was prepared. The azidosilane solution was spin
coated onto the silicon wafer by placing a 300 .mu.l sample of the
azido-silane solution in the center of the silicon wafer while the
wafer was spinning at 5,000 rpm in a dry N.sub.2 environment. The
azido-silane coated wafer was then spun dry, spin rinsed with 1 mL
of 200 proof EtOH, and baked at 110.degree. C. for 10 minutes.
[0079] A 0.5% 212,000 MW styrene (30-33 mV) in toluene solution was
prepared. The styrene solution was spin coated onto the
azido-silane layer by placing a 500 .mu.l sample of the styrene
solution in the center of the azidosilane coated wafer while the
wafer was spinning at 5,000 rpm in an ambient environment. The dual
element attachment layer and wafer were then baked at 100.degree.
C. for 18 to 20 hours and the attachment layer was then checked for
attachment stability.
EXAMPLE 2
[0080] Rabbit anti-Bacillus globigii polyclonal antibodies are
diluted to 9 .mu.g/mL in 50 mM HEPES buffer (pH 8.0), and 15 .mu.L
of this diluted capture antibody are dispensed onto each sensing
unit in the test piece. The test piece is then incubated for one
hour at 37.degree. C. under high humidity conditions. After rinsing
with distilled water and drying the test piece with compressed
nitrogen gas, any additional protein binding sites in the sensing
units are blocked by adding 18 .mu.L StabilCoat.TM. to each sensing
unit. After incubation for one hour at 37.degree. C., excess liquid
is removed by aspiration, and the test piece may be stored at
4.degree. C. until use.
[0081] When the assay is to be carried out, the excess blocking
protein is rinsed away using distilled water. Fifteen microliters
of sample (diluted into Bacillus globigii Sample Buffer consisting
of 50 mM HEPES, pH 8.0, with 0.2% bovine serum albumin, 0.01%
Tween.RTM.-20) are dispensed into the sensing units, and the test
piece is incubated at 37.degree. C. for one hour. The sensing units
are then rinsed with distilled water and dried with compressed
nitrogen gas.
[0082] In the next step, 15 .mu.L biotinylated goat anti-Bacillus
globigii polyclonal antibodies (diluted to 2 .mu.g/mL in
anti-Bacillus globigii Sample Buffer) are dispensed into each
sensing unit. The test piece is incubated for thirty minutes at
37.degree. C. The reaction is stopped by rinsing the test piece
with distilled water and drying with compressed nitrogen gas. Next,
10 .mu.L streptavidin/polymerized horseradish peroxidase conjugate
(Sigma Chemical Company; St. Louis, Mo.), diluted 1:6000 in
Bacillus globigii Sample Buffer, are added. The test piece is
incubated for thirty minutes at 37.degree. C., and the test piece
is then rinsed with distilled water and dried with compressed
nitrogen gas.
[0083] Finally, 10 .mu.L TMB Reaction Substrate (Kirkegaard Perry
Laboratories; Gaithersburg, Md.) are added to each sensing unit.
After incubation for 15 minutes at 37.degree. C., the reaction is
stopped by rinsing the test piece with distilled water. The test
piece is dried with compressed nitrogen gas, and the amount of
reaction product is measured on the instrument.
EXAMPLE 3
[0084] Sensing units in the test piece are coated with 15 .mu.L of
rabbit anti-Listeria polyclonal antibodies, diluted to 23 .mu.g/mL
in PBS (10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl).
After incubating the test piece for one hour at 37.degree. C. under
high humidity conditions, the test piece is rinsed with PBST (PBS
containing 0.05% Tween.RTM.-20), following by rinsing with
distilled water. The test piece is then dried with compressed
nitrogen gas. Any additional protein binding sites are blocked by
adding 20 .mu.L bovine serum albumin, 30 mg/mL in PBS, to each
sensing unit. After incubation for 30 minutes at 37.degree. C.,
excess liquid is removed by aspiration, and the test piece may be
stored at 4.degree. C. until use.
[0085] In the next step, 15 .mu.L sample, consisting of heat-killed
Listeria in media, is added to the sensing units. The test piece is
incubated for 30 minutes at 37.degree. C., and then rinsed with
PBST and distilled water. The test piece is dried with compressed
nitrogen gas. Next, 15 .mu.L rabbit anti-Listeria polyclonal
antibody/horseradish peroxidase conjugate (Kirkegaard Perry
Laboratories), diluted 1:500 in Biostride.TM. conjugate diluent,
are added to each sensing unit. The test piece is incubated for 30
minutes at 37.degree. C., rinsed with PBST and distilled water, and
then dried with compressed nitrogen gas.
[0086] Finally, 15 .mu.L TMB Reaction Substrate (Kirkegaard Perry
Laboratories) are added to each test well. After incubation for 15
minutes at 25.degree. C., the reaction is stopped by rinsing the
test piece with distilled water. The test piece is dried with
compressed nitrogen gas, and the amount of reaction product is
measured on the instrument.
Instrument and System Operation
[0087] With reference to FIGS. 9 and 10, the system for detecting a
specific substance or analyte of interest also includes an
instrument 300 for obtaining and analyzing data related to the
determination of whether or not the analyte of interest is present
with a sensing unit 100. The instrument 300 includes a housing 304
for containing assemblies and components utilized in the detection
and analysis process. The housing 304 is characterized by its
compact size and relatively small footprint. Its compact size is
achieved by specific selection and arrangement of such assemblies
and components within the housing 304. The housing 304 includes a
lower containing unit 308 having a number of walls and an upper
cover 312 that is connected to the lower containing unit 308. The
cover 312 has an upper face that includes an input unit or keyboard
316 by which the technician or user can input information including
requests for data to the instrument 300. The upper face also has a
window 320 for receiving a liquid crystal display (LCD) 352 (FIG.
10) that is useful in displaying menus or other information for
selection, together with results of the analysis conducted by the
instrument 300, such as graphic plots of data related to the
detection process. An end wall 324 of the lower housing unit 304
has a receiver slot 328 formed near the bottom of this end wall
324. The receiver slot 328 is configured and of a size to receive a
test piece or slide 332. As seen in FIG. 11, the test piece 332 is
an elongated, relatively flat member that is able to hold a number
of sensing units 100, each of which is spaced from any other such
unit 100. The test piece 332 has indicia 334 for indicating the
location and/or identity of the particular sensing unit 300. That
is, such indicia 334 may include one or more marks associated with
or located at each sensing unit 100a-100l. Consequently, when such
a mark is read or detected by the instrument 300, this read
information can be used to initiate the analysis and detection
process. Additionally or alternatively, such indicia 334 may
include identification information, such as in the form of a bar
code associated with or located adjacent to each sensing unit
100a-100l. The bar code for each sensing unit represents the
identity of the particular sensing unit 100a-100l. Accordingly,
such bar code information can be used to maintain or record
identity information for the particular sensing unit that is being
tested and distinguish it from other sensing units.
[0088] Returning to FIGS. 9 and 10, one of the assemblies of the
instrument 300 is a controller assembly 340. The controller
assembly 340 has a number of electronic components, including a
digital controller 344 that is responsible for controlling and
interacting with a number of devices or elements. The digital
controller 344 includes one or more processors that are involved
with determining whether or not a specific analyte of interest is
present in the sensing unit 100 being tested using data that the
processor receives from other components of the controller assembly
340. The digital controller 344 communicates with a LCD controller
348, which is used in controlling and providing the presentation of
information by the liquid crystal display (LCD) 352, under the
ultimate control of the digital controller 344. Similarly, the
digital controller 344 communicates with the keypad 316 through a
keypad interface 356 before sending such information to the digital
controller 344. The controller assembly 340 also includes memory
storage for storing data and executable code. In that regard, a
program memory 360 and a data memory 364 communicate with the
digital controller 344. The program memory 360 stores programmed
code or algorithms for analyzing the data-related signals that are
received by the digital controller 344, including those related to
detecting the presence of an analyte of interest. The data memory
364 is utilized to store results associated with the detection
process, such as values related to the results of the detection
process.
[0089] Returning to FIG. 9, the components and assemblies of the
controller assembly 340 are mounted on one or more printed circuit
boards 368 that are sized to be contained within portions of the
lower containing unit 304. Also included within the lower
containing unit 304 and in communication with the digital
controller 344 is a number of data providing/generating or
peripheral assemblies, with at least portions thereof disposed
below the printed circuit board(s) 368. More specifically, a test
piece control assembly 372, a reader assembly 376 and a light bean
assembly 380 are provided, with at least some elements of each of
these three assemblies being fixed to a mounting plate 384.
[0090] With reference to FIGS. 12 and 13, as well as FIGS. 9 and
10, each of these assemblies will be described in greater detail.
The test piece control assembly 372 is employed in connection with
controlling movement of the test piece 332 having a number of
sensing units 100 that may have an analyte of interest. The test
piece control assembly 372 includes a motor 388 that is held or
supported by a motor mount 392. Operation of the motor 388
including providing and removing power thereto is accomplished
using a motor controller 396 that communicates with the digital
controller 344. When it is appropriate to move the test piece 332,
the digital controller 344 generates the necessary signal for
receipt by the motor controller 396 in order to generate a suitable
signal for the motor 388. The output of the motor 388 is connected
to a first gear 400 that rotates when the motor 388 is powered. The
first gear 400 operatively engages the second gear 404. The two
gears 400, 404 are at right angles to each other to contribute to
the compact size of the instrument 300. Rotation of the first gear
400 causes the second gear to also rotate. The second gear 404 is
connected to a drive shaft 408 having a first collar 412 adjacent
to the second gear 404 and a first bushing 416 spaced from the
collar 412 along the drive shaft 408. First and second support arms
420, 424 receive and support the drive shaft 408. Between the
support arms 420, 424, the shaft 408 traverses a test piece path
428 defined in the mounting plate 384. The first and second support
arms 420, 424 are held to the mounting plate 384 on opposite sides
of the test piece path 428. Inwardly adjacent to each of the two
support arms 420, 424 is first drive wheel 432 and second drive
wheel 436, respectively. Each of these two drive wheels 432, 436 is
toothed or otherwise configured to satisfactorily engage edges or
other portions of the test piece 332. Each of the two drive wheels
432, 436, through engagement with the drive shaft 408, rotates when
the drive shaft 408 is driven or rotated to thereby cause the test
piece 332 to move along the test piece path 428. To complete the
discussion of the elements supporting the drive shaft 408, a second
bushing 440 is provided about the shaft 408 adjacent to the second
drive wheel 436 and a second collar 444 is disposed about the drive
shaft 408 on the opposite side of the second support arm 424. The
test piece control assembly 372, as seen in FIG. 4, also includes a
back plate 448 having an acceptor opening 452 oriented with the
receiver slot 328 formed in the end wall 324 of the lower
containing unit 308 in order to receive and guide the test piece
332 along the test piece path 428.
[0091] With regard to positioning the test piece 332 at a desired
position for a particular sensing unit 100 to be analyzed, the
reader assembly 376 is utilized. The reader assembly 376 reads or
obtains information using the indicia 334 on the test piece 332.
This obtained data is sent to the digital controller 344, which
analyzes such information in controlling the application of power
to the motor 388. As seen in FIGS. 12 and 13, the reader assembly
376 includes a reader element 460 that is precisely and accurately
positioned along the test piece path 428 to read indicia 334 on the
test piece 332. The position of the reader element 460 is precisely
positioned relative to certain elements of the light beam assembly
380, as will be understood more fully when a more detailed
description of the light beam assembly 380 is provided. The reader
element 460 communicates its output to processing circuitry 464 of
the controller assembly 340, as illustrated in FIG. 9. The
processing circuitry 464 processes the signal received from the
reader element 460 to output a reader signal, including providing
desired amplification, filtering to obtain the proper frequency for
the signal and removing unwanted noise from the signal. When a
modulated light source is utilized, filtering that enhances the
ability to differentiate the desired signal from an unwanted DC
level of noise is provided. In such a case, such filtering can
include the use of Fourier transform techniques that are useful in
extracting the DC noise portion from the input signal, then
applying an electronic filter and subsequently integrating the
resultant signal to obtain the desired, detected signal. After such
processing, the processed reader signal is applied to a decoder
468. In the case of reading a bar code, the decoder 468 includes
logic for deciphering or otherwise analyzing the signal input
thereto in order to generate a digital signal that is indicative of
the bar code currently read by the reader element 460. This digital
or decoded signal is applied to a serial interface 472 that
communicates with the digital controller 344 to thereby provide
test piece 332 position information to the digital controller
344.
[0092] Returning to FIG. 12, the reader assembly 376 also includes
a reader mount 476 having a hole for receiving and tightly engaging
the reader element 460. The reader mount 476 is adjustable relative
to the test piece path 428 in order to properly align it relative
to the test piece path 428. In that regard, the reader assembly 376
also includes an adjusting element 480 located in an adjusting slot
484 whereby the reader mount 476 can be variably located relative
to a reader support 488 that is fixably held to the mounting plate
384 adjacent to the test piece path 428.
[0093] The light beam assembly 380 also has elements that must be
accurately and precisely located relative to the test piece path
428. The light beam assembly 380 provides a light beam that is used
in illuminating the sensor containing the analyte of interest, when
present, on the particular sensing unit 100 when the test piece 332
is controllably stopped along the test piece path 428 in order to
enable the light beam to strike and reflect from the sensing unit
100. In the embodiment of FIGS. 9, 10, 12 and 13, the light beam
assembly 380 includes a laser module 496 that provides a source of
monochromatic light, although a non-laser source of generated light
is feasible. The monochromatic light source may include properly
filtered white light, a light emitting diode (LED) or a laser
diode. The application of power and the control associated with the
light beam outputted by the laser module 496 is provided using the
digital controller 344 and a laser regulator 500, which
communicates with the digital controller 344. As illustrated in
FIGS. 7 and 8, the laser module 496 is supported in an optics mount
504. In the embodiment of FIG. 8, the light beam assembly 380 also
includes an adjustable or rotatable linear polarizer 508 for
desirably preparing the polarization state of the light beam that
is to be incident upon the sensing unit 100 after passing through a
polarizer plate 512. More specifically, the linear polarizer 508 is
orientated to output only the "s" component or "s" polarization of
the light it receives while essentially eliminating or minimizing
the "p" component or p-polarization of the light it receives. In
another embodiment, a wave plate or compensator is also used in
combination with the polarizer 508 prior to the light striking the
sensing unit 100 in order to assist in achieving the desired
polarized state of the light. In other embodiments, circular or
elliptical states of polarized light are obtained and utilized.
[0094] The incident linearly polarized light from the polarizer
plate 512 is directed to the particular sensing unit 100. The
incident light strikes a small area or point on the sensing unit
including the analyte of interest, when present. The reflected
light includes useful information in connection with determining a
change in mass when the specific analyte of interest is present.
The light beam assembly 380 also includes a detection assembly 520
that is properly positioned and aligned to receive such reflected
light. As long as metal reflectors or materials that have a large
imaginary component (e.g., greater than 0.03) of index of
refraction are not being used as part of the sensing unit 100,
mostly linearly polarized light (s-polarization only) will leave
the sensing unit 100 after reflection.
[0095] In other embodiments, the light beam assembly 380 is
configured to result in only p-polarized light after reflection. An
elliptical state of the polarized light can also be achieved in
which the elliptical polarized light strikes the sensing unit 100
and the resulting polarized state of reflected light is also
elliptical. Alternatively, circular polarized light may be
utilized. However, the circular polarization may undergo an
interaction at the sensing unit 100 that changes the polarized
state of the reflected light to be slightly elliptical.
[0096] The detection assembly 520 includes a detector polarizer 528
that removes, extinguishes, filters or absorbs the linearly
polarized light that it receives. In another embodiment, a wave or
compensator plate may be optionally used with the polarizer 528.
Such a component assists in filtering the reflected state of
polarized light of any small component of p- or s-polarized light
provided in the reflection from the sensing unit 100. It is
possible that the composition of the sensing unit 100 may cause
some ellipticity to be present in the reflected light and/or that
the ellipticity is caused by slight imperfections or small
misalignments of the polarizing components. After the foregoing
optical processing, the only signal produced, in view of the
removal of the linearly polarized light by the detector polarizer
528, will be due to an increase or decrease in the mass of the
sensing unit 100. It has been observed that this arrangement
decreases the sensitivity to small increases in sensing unit mass
changes, with the sensitivity being mainly due to the use of only
s-polarized light. On the other hand, a decrease in sensitivity is
experienced when other than p- or s-polarized light enters the
detector polarizer 528. If any p-polarized light were present,
there would be a lower signal-to-noise ratio, which means less
sensitivity to small mass changes. More particularly, if the
imaginary component of the index of refraction of the sensing unit
100 is at least 0.05, then the light leaving the sensing unit will
be elliptically polarized. Elliptical polarization has both "s" and
"p" components present. When both of the components are present,
the light cannot be extinguished by the detector polarizer 528. In
the embodiment being described, the s-polarized light is
extinguished and the p-polarized light would continue along the
optical detection path. This p-polarized light constitutes optical
noise and decreases the sensitivity of the instrument.
[0097] In the embodiment of FIGS. 12 and 13, the detection assembly
520 also includes a compensator wave plate 524 that is optically
positioned between the sensing unit 100 and the detector polarizer
528. The compensator wave plate 524, typically a quarter-wave
plate, allows elliptically polarized light that might leave the
sensing unit 100 to be converted to plane or linearly polarized
light having only the "s" component. When linearly polarized light
is present, it can then be extinguished by the detector polarizer
528 prior to detection of any light signal that might be generated
due to a mass or thickness change in the sensing unit 100.
[0098] It should be appreciated that the wave or compensator plate
previously discussed can be used on either side of the reflection
from the sensing unit 100. The choice of retardation in the
compensator plate is based on what is required to optimize the
signal-to-noise ratio of the particular optical component
configuration. Depending upon such circumstances, a 1/8, 1/4, 1/2
or other wave retardation can be selected.
[0099] In another embodiment, instead of s-polarized light being
applied to the detector polarizer 528 to be extinguished by it,
only p-polarized light is produced and applied to the detector,
with the "s" component polarizer 528 already having been
essentially eliminated or minimized by the polarizer elements that
output the incident light to the sensing unit 100.
[0100] The output of the detector polarizer 528 is applied to a
detector unit 532 that includes, for example, a photodiode for use
in detecting or measuring the intensity of the light received by
it. The intensity of the light detected relates to the change in
mass due to the analyte of interest when it is present with the
sensing unit 100 as will be described further herein. As seen in
FIG. 10, the detector unit 532 communicates with detector
processing circuitry 536 of the controller assembly 340 (FIG. 9).
The detector processing circuitry 536 processes the analog signal
from the light detector unit 532 including appropriate
amplification and signal filtering including, as previously noted,
the use of Fourier transform filtering. The output of the detector
processing circuitry 536 is applied to an analog-to-digital
converter (ADC) 540 for converting the analog signal to a digital
signal acceptable to the digital controller 344. This digitized
light signal represents the light intensity of the reflected light
from the sensing unit 100, including analyte of interest, when
present. This digital light signal is then analyzed in connection
with making a determination regarding the presence or absence of
the analyte of interest with the particular sensing unit 100 being
tested.
[0101] In another embodiment, instead of a photodiode type of
detection, an imaging detector, such as a video type, charge
coupled device (CCD) and so forth, can be used to capture intensity
change of the sensing unit as well as an image of the sensing unit
being tested. This type of image inspection would be equivalent to
scanning the sensing unit with a 0.012 mm laser beam. Using data
from about 0.012 mm section of a sensing unit, different types of
digital analysis can be applied to determine the amount of material
captured. For instance, if some non-specific material were also
captured with the analyte of interest, and they were larger in size
or had different polarization properties, their presence could be
detected when their size is in some reasonable relation to the
0.012 mm detection resolution, or the polarization property were
high enough above general noise. This means that these areas are
potentially selectable and can be eliminated from the overall
signal.
[0102] In another embodiment, an optical apparatus involved with
the control of the incident light and collection of reflected light
utilizes two detectors and no compensator plate. Such a
configuration does not contain any moving or adjustable components
like polarizers or compensator plates. In accordance with this
configuration, light will reflect from the sensing unit at the
proper or desired angle other than 0.degree., 90.degree. or
Brewster's angle. After leaving the sensing unit, the light will
again reflect from a detector or silicon substrate that is
positioned at or near Brewster's angle. This reflection will
eliminate all of the p-polarized light and leave only s-polarized
light. The use of a silicon detector for the reflected light after
the sensing unit is beneficial since a reading of the amount of
p-polarized light can be obtained, which may prove valuable in
making precise calculations regarding the material on the surface
of the sensing unit. The remainder of the reflected light will be
collected by a final or another detector, comparable to those
previously discussed. The final value of the s-polarization light
that was collected can be compared to s-polarization light that is
collected from a sensing unit that has no analyte of interest. An
instrument that includes this optical apparatus can have three
outputs, s-polarized light, p-polarized light and the ratio of the
p-to-s amplitudes. This ratio is a common ellipsometric calculation
and can assist calculating other values of interest.
[0103] Additional details of the embodiment of FIGS. 12 and 13
associated with the analysis are next described with reference to
the flow diagram of FIG. 14. In accordance with the method of this
embodiment, unlike known prior art, only one polarized light
component is received at the detector polarizer 528, such as the
s-polarization, while the linear polarizer elements 508, 512 and
the detector polarizer 528 remain stationary in their same position
or orientation that each had when the incident light is first
generated and then directed to the particular sensing unit 100 that
is being tested.
[0104] With respect to using the determined light intensity from
the sensing unit 100 in order to detect whether or not the analyte
of interest is present, the present invention relies on previously
generated standardized data that may be presented in the form of
standardized curves or plots. Such standardized data may also be
represented by a number of discrete data points stored in memory
that can be interpolated to arrive at any value between such
discrete data points. Such data points relate to values of light
intensity associated with a number of standardized or accurately
measured masses for a number of sensing units that include masses
that are intended to correlate with changes in mass to a sensing
unit currently being tested when a substance of interest is
present. For example, the instrument 300 is used with a
standardized sensing unit 100 that includes a mass representative
of a mass when an analyte of interest is present. Measurements are
conducted using this standardized sensing unit. Data is collected
for this particular sensing unit having this first known mass, as
well as other sensing units having other known masses. Such known
masses are correlated with light intensity values that are
obtained. The results of such standardization development include
correlated masses and light intensity values, as noted in step 550
of FIG. 14.
[0105] With such information or data available, step 554 defines a
calibrating step that is conducted prior to performing the
detection process associated with a particular sensing unit 100.
Specifically, the detector linear polarizer 528 is calibrated by
desired rotation thereof to obtain a reference light intensity that
passes through it when a known or standard sensing unit is present
to reflect light. This reference light intensity may have a zero or
substantially zero intensity value. After this calibrating step
that involves rotating the detector linear polarizer 528, at step
558, a sensing unit 100 is then properly positioned to receive
incident light. At step 562, the incident light is directed to a
point or small area on the particular sensing unit 100. Reflected
light from this point on the sensing unit 100, including analyte of
interest when present, is received through the compensator wave
plate 524, in accordance with step 566. At step 570, the reflected
linearly polarized light is received through the detector polarizer
528, which is not rotated during this detection process but remains
stationary, as recited in step 574. After the intensity of the
light is obtained using the light detector unit 532, the detector
circuitry 536 and the analog-to-digital circuitry 540, the digital
light signal representative of the intensity is applied to the
digital controller 344. At step 578, the digital controller 344
including a processor thereof compares the digital light signal
with the previously developed data, as noted in the discussion of
step 550. Based on a comparison between the previously developed
standard data and the obtained light signal, a determination is
made as to any change in mass in the sensing unit 100. Based on
such a determination, a result can be provided as to whether or not
the analyte of interest is present.
[0106] The result of this determination can be displayed using the
liquid crystal display 352. Additionally or alternatively, such
result information, as well as other data, can be supplied or
downloaded to an external apparatus, such as a computer system
using a serial I/O connection 590 of FIG. 10 that communicates with
the serial interface 472 to the digital controller 344. The data
associated with the result of each such test can also be stored in
the data memory 364 for later access and use.
[0107] Regarding data that is obtainable in connection with
analysis of one or more sensing units 100, a further description is
provided with reference to FIGS. 15-16. In accordance with this
method, a differential analysis is conducted. More specifically, a
difference in results is obtained between related sensing units 100
found on successively tested test pieces. That is, a difference is
taken between the results obtained for a first sensing unit, which
is being tested for an analyte of interest, and a sensing unit that
does not have such an analyte of interest. The first sensing unit
is positioned on a first test piece and the second sensing unit is
positioned on a second test piece at a corresponding location. The
difference that is found is a signal or light intensity due to the
analyte of interest when present from the chemical reaction or
capture process. These operational steps also differ from known
prior art in connection with the use of a controllable motor for
automatically positioning each of a number of sensing units on a
test piece relative to components that are involved in the
detection process.
[0108] FIGS. 15A-15B illustrate flow diagrams setting out major
steps in conjunction with obtaining results and associated data for
a number of sensing units 100 on a first test piece 332 for use in
determining whether one or more of them has an analyte of interest.
In accordance with step 600 of FIG. 15A, a first test piece 332
having a number of sensing units is positioned for movement
relative to the instrument 300. At step 604, the first test piece
332 is moved relative to the instrument 300 along the test piece
path 428. A continuous check is made at step 608 for a mark and/or
identification code (bar code) or any other indicia on the first
test piece indicative of a first sensing unit 100. At step 612,
movement of the first test piece 332, under control of the test
piece control assembly 372 and the controller assembly 340, is
discontinued, based on a determination using the reader assembly
376 that the first sensing unit 100 on the first test piece is
properly positioned for analysis, including the obtaining of a
reading related to whether or not an analyte of interest is present
with this first sensing unit 100. A desired position of the test
piece 332 along the test piece path 428 for conducting the test on
the first sensing unit 100a is illustrated in FIG. 16.
Subsequently, the light beam assembly 380 is activated to take a
reading and conduct the analysis for this first sensing unit of the
first test piece at step 616. The reading obtained is indicative of
the presence or absence of the analyte of interest for this
particular sensing unit 100 and at step 620, the results and
associated information for this first sensing unit on the first
test piece 332 is stored in the data memory 364. In one embodiment
of the invention, the data that is stored includes a location
number indicative of the position or number of the sensing unit 100
for the first test piece 332, an identification code that
identifies the sensing unit, the result of the analysis in terms of
a quantitative value obtained using the digital light signal
generated using the light beam assembly 380 and subsequent
processing circuitry, the date that the analysis was conducted and
the time of such analysis. In accordance with step 624, steps
604-620 are repeated for each sensing unit 100 that is found with
the first test piece 332. Then a second test piece 332 having a
corresponding number of sensing units 100 is provided, with each of
these not having an analyte of interest. Steps 600-624 are
conducted using this second test piece 332 in accordance with step
628. With respect to the results that are obtained using the
digital light signal, a difference is taken between corresponding
sensing units of the first and second test pieces when there are
matching identification codes at step 632 of FIG. 15B. For example,
the determined result of the analysis for location 1 of the first
test piece is subtracted from the determined result for location 1
of the second test piece when the identification codes match and
these two locations correspond to each other. As noted, any
sufficient difference is indicative of a mass or thickness change
and, concomitantly, an indication that an analyte of interest is
present with the sample on the sensing unit 100 that was tested. At
step 636, the result of each of the subtractions is saved or stored
and, if the result is less than zero, the value 0.0 is stored. At
step 640, on the other hand, if it is found that there is a lack of
correspondence between the first and second test pieces, no
subtraction is taken and no readings are stored.
[0109] The program code or software that is useful in implementing
the foregoing steps also includes a number of capabilities, which
are identified by steps 644-652 of FIG. 15B. At step 644, a recall
menu is displayed using the liquid crystal display 352. A number of
recall functions are available for implementation. "Recall Last",
when invoked or pressed, displays information about the last test
that was conducted and the reading taken including displaying
identification information, the date and time that the reading was
obtained, together with the reading result that, in one embodiment,
is displayed in volts. "Recall/Scroll", when invoked or pressed,
displays the same results as "Recall Last" and scroll keys are used
to enable the user to scroll over and back through all memory
locations. "Recall By ID", when invoked or pressed, allows the user
to enter the identification code and information is displayed
relating to all readings that have that identification code.
[0110] At step 648, the user is able to enter a "Plot Request" by
which a number of samples of data for a single identification code
are displayed. In order to make this request, the desired
identification code is entered and an initial number of readings,
up to a maximum number, is plotted and displayed. When there are
more than the maximum number of readings for the entered ID, the
additional or excess samples can also be plotted by pressing an
appropriate key on the keypad 316. When there is no match between
the requested identification code and available readings, a display
is provided to the effect that no such readings are available for
that entered identification code.
[0111] In accordance with step 652, the program code includes a
number of utilities that can be accessed. Display 352 is used to
provide the utilities menu or menus. The utilities menu includes
"Show System" for displaying current information related to the
particular instrument 300 including: the current memory location
for the sensing unit under test; the total number of memory
locations for the particular instrument; the number of memory
locations still available; the current date; the current time; the
current identification code; the number of locations that will be
read associated with a test piece having the sensing units; the
current gain for the detector processing circuitry 536; the current
software version and the current hardware version. The utilities
menu also includes a "download data" which is used to download all
current data stored in the data memory 364. The downloaded data can
be received by, for example, a personal computer. The utilities
menu also includes the following: "change identification code" by
which the user is able to change the current identification number
or code to another number or code; a "change gain" by which the
user is able to adjust the gain between minimum and maximum values;
"change number of locations" by which the user is able to adjust
the number of locations on the test piece 332 that will be
automatically read; "laser on/off" by which the power to the laser
can be separately controlled; and "motor CW/CCW" by which a key is
used to turn on the motor 388 and toggle the direction of the motor
in connection with confirming the current direction thereof.
Numerous other utilities can be provided or implemented depending
upon the needs of the user.
[0112] In another embodiment of an instrument primarily
characterized by a differently configured test piece control
assembly 660, reference is made to FIGS. 17-20. The test piece
control assembly 660 for moving the test piece 332 includes first
and second drive rollers 662a, 662b. A drive plate 664 is
operatively connected to each of the two drive rollers 662a, 662b.
A continuous conveyor belt 668 is disposed over the drive rollers
662a, 662b, as well as the drive plate 664. The test piece control
assembly 660 also includes a guide assembly 670 that is useful in
properly locating or guiding the test piece 332 during its
controlled movement along and on top of the conveyor belt 668. In
that regard, the drive roller 662a preferably has a biasing
mechanism, such as a spring, that causes desired movement in a
direction toward the guide assembly 670 to provide suitable
positioning of the test piece 332 relative to the guide assembly
670. More specifically, the guide assembly 670 includes a number of
guide rollers 674 that are spaced along the length of the conveyor
belt 668. In the illustrated embodiment, four such guide rollers
674a-674d are provided. Each of the free ends of the guide rollers
674 engages the edge of the test piece 332. The guide assembly 670
also includes a pair a pawls 680a, 680b that are located adjacent
to the entry end of the test piece 332 onto the conveyor belt 668.
Each of the two pawls 680a, 680b is desirably positioned for
guiding the test piece 332 relative to the top of the conveyor belt
668 as the test piece 332 is received. The test piece control
assembly 660 also includes first and second support walls 682a,
682b that are spaced from each other and have the drive rollers
662a, 662b, together with the drive plate 664 and the conveyor belt
668, positioned therebetween. The support wall 682a supports the
guide rollers 674 and the pawls 680 relative to the conveyor belt
668 and the test piece 332 when it is present in the instrument. As
illustrated in FIG. 17, the embodiment of FIGS. 17-20 also includes
the support wall adjustment assembly 684. This adjustment assembly
684 is operably connected to the second support wall 682b and is
used in moving or adjusting the position of the second support wall
682b relative to the first support wall 682a. Accordingly, the
spacing or distance between the two walls 682a, 682b can be varied
in accordance with the width of the test piece 332. In this way, an
optimum spacing is achieved that is a function of the width of the
test piece 332 so that the test piece 332 can be properly located
and guided between the support walls 682a, 682b. As further denoted
in FIG. 17, this embodiment also includes a light beam adjustment
assembly 686 that includes an alignment member 688 and a number of
fasteners 690 joined thereto. The fasteners 690 are operably
connected to the laser module 496 of the light beam assembly 380.
Optimum positioning of the laser module 496 and, concomitantly,
optimum directing of the light beam outputted therefrom is achieved
by adjustment of the position of the laser module 496 using the
fasteners 690. By this arrangement, instead of time-consuming and
potentially imprecise locating of the laser module 496 in the
instrument, adjustment to obtain its optimum position is achieved
by movement of the fasteners 690 causing desired movement of the
laser module 496 until the optimum position is found. With
reference to FIG. 20, a housing 692 for this embodiment is
illustrated. The housing 692 includes an angled receiver slot 694
that is geometrically designed to facilitate the entry of a test
piece 332 so that it is properly guided for receipt by the test
piece control assembly 660. The housing 692 also has a pair of
cut-outs 696a, 696b that permits manual access and movement of the
test piece relative to the mechanisms and components located within
the housing 692.
Additive Polarization Optics
[0113] Other embodiments of the assemblies and components of the
instrument 300 can be utilized. With regard to another embodiment
of a light beam assembly, reference is made to FIGS. 21 and 22.
This embodiment incorporates a "multi-bounce" technique by which
the sensing unit 100 being tested is subject to a number of
reflected light passes, instead of only one. If an extremely small
light beam were used as part of the multi-bounce operation, it
could be concluded that the light beam would reflect from a
different spot on the sensing unit 100 on each pass of the light.
However, the light beam commonly has some finite size, and the area
occupied by the analyte of interest, when present, is typically
very small so that it is reasonably concluded that the light beam
reflects from a small area on the sensing unit and successive
reflections will have some overlap with one or more previous small
areas that were contacted by the light beam. Such a process of
overlapping reflections assists in "averaging out" the total
surface area of the sensing unit.
[0114] In utilizing the "multi-bounce" technique, the main
objective is to produce a sufficient signal above the background
noise or other signals to be detected. One bounce or reflection
from the sensing unit 100 may not be sufficient to produce such a
signal. In order to detect a very thin change in thickness or small
change in mass associated with the sensing unit 100 when the
analyte of interest is present, a signal produced by one bounce or
reflection may not be enough. By causing more reflections on passes
of light through the sensing unit and adding each small change of
polarization to the previous changes (additive or cumulative
polarization per reflection on the sensing Unit with analyte of
interest when present), then eventually the change in polarization
will produce a sufficient intensity change, as represented by the
resulting signal, so as to be detected. The multi-bounce embodiment
differs from known prior art in generating multiple reflected
signals that are at angles other than zero degrees, preferably also
different from 90.degree., to the sensing unit and collecting at
least portions of such reflected light signals and analyzing the
total collected light signal in determining whether an analyte of
interest is present.
[0115] With reference to FIG. 21, an embodiment of a light beam
assembly 700 is schematically illustrated that generates multiple
reflections and uses partial light transmissions to analyze the
intensities of the collection of such partial transmissions. More
specifically, the light beam assembly 700 includes a polarized
light source 704 that may be monochromatic or some other acceptable
source. A curved mirror 708 is properly disposed in the path of the
light beam from the source 704. The mirror 708 has optical power
and special reflective coatings. A quarter wave retarder or
compensator plate 712 is positioned in the path of the light beam
that exits the curved mirror 708. The quarter wave retarder plate
712 has an anti-reflective coating on both of its sides. The plate
712 is disposed between the curved mirror 708 and a sensing unit
100. The function of the quarter wave retarder plate 712 is to
cause the polarization state that passes through it to be additive
after reflection, and not cause a cancellation of the polarization
change, as will be explained later herein. The light beam assembly
700 also includes a focusing lens/analyzer plate 716 that is in the
path of the partially transmitted light from the curved mirror 708.
The focusing lens/analyzer plate 716 can be rotated about its axis
in connection with controlling the receipt of the transmitted light
from the curved mirror 708. The focusing lens/analyzer plate 716 is
suitably positioned to direct the partially transmitted light from
it to a detector unit 720, which collects all of the partially
transmitted light for subsequent conversion to a light signal
representative of the intensities of the collected light. This
light signal is analyzed, similar to the embodiment previously
described, in connection with determining any change in mass or
thickness of the sensing unit 100 due to the presence of an analyte
of interest thereon.
[0116] With respect to a discussion of the number of "bounces" or
reflections, as FIG. 21 illustrates, the polarization light source
704 directs the light beam to the curved mirror 708. Light from the
source 704 may or may not pass through the curved mirror 708 and is
incident upon the sensing unit 100 at a first point or spot. Light
is reflected therefrom and contacts the curved mirror 708 whereby
there is a partial transmission of light A together with a further
reflection from the curved mirror 708 to a different point on the
sensing unit 100. This results in a reflection back to the curved
mirror 708, where there is a partial transmission of light B,
together with another reflection back to the sensing unit 100. Yet
another reflection occurs from the sensing unit 100 at a different
point thereon to the curved mirror 708, where partially transmitted
light C passes through the curved mirror 708, while additionally
reflected light from the curved mirror 708 is directed back to the
sensing unit 100 at a different point. At this further different
point, another reflection occurs from the sensing unit 100 to the
curved mirror 708 and yet another partial transmission light D
occurs.
[0117] For each of the reflections between the curved mirror 708
and the sensing unit 100, such light passes through the quarter
wave retarder plate 712. Each time there is a pass through the
plate 712, the polarization state of the light is 90.degree. plus a
small change that occurs due to the thickness of the sensing unit
100. The curved mirror 708, on the other hand, has special optical
properties regarding polarization and will not introduce any
additional polarization and is termed a neutral polarization
reflector. Consequently, the only polarization change due to the
curved mirror 708 is the 180.degree. change due to the reflection.
On the other hand, the quarter wave retarder plate 712 causes the
polarization vector to point in the opposite direction from the
direction it had when it entered the quarter wave retarder plate
712. After two passes through the retarder plate 712 and one
reflection, there is a 360.degree. rotation in the polarization
state, together with a small change due to the thickness of the
sensing unit 100. In the absence of the retarder plate 712, the
vector sum of the polarizations at the sensing unit would be
180.degree. out of phase and would cause a cancellation of the
phase change gained on the reflection at the sensing unit. However,
by including the one quarter wave retarder plate 712 to the optical
path, it produces two 90.degree. polarization changes, each time
light passes through first one side thereof and then through the
second side thereof. This summing of polarization changes continues
for each of the reflections.
[0118] Regarding the generation of the partially transmitted light
and the multiple reflections of the entering light, this is
achieved by the optical power or curvature of the curved mirror
surface 708 and the entering angle of incidence of the light beam
from the source 704. The reflective properties of the curved mirror
708 also allow for the partial transmission of light through the
curved mirror 708 each time reflected light strikes the curved
mirror 708.
[0119] A further description of this embodiment is provided with
reference to the flow diagram of FIG. 22 that is directed to major
steps related to the obtaining and analyzing of data related to
light intensities for determining mass changes. In particular, step
750 of FIG. 22 involves the development of standardized data using
a number of standard masses and measuring of light intensities,
similar to the step of 550 of FIG. 14 in the previously described
embodiment. In accordance with step 754, the focusing lens/analyzer
plate 716 must be properly oriented for collection of the numerous
partial light transmissions through the curved mirror 708. In that
regard, the necessary orientation is previously determined and
based on the anticipated masses of the sensing units 100. That is,
in connection with testing for a specific analyte of interest, the
expected mass range for this analyte of interest was previously
determined, and the focusing lens/analyzer 716 is oriented at an
angle based on this previous determination. At step 758, power is
applied to the light beam source 704, which directs the light beam
to the curved mirror 708. A number of reflections are generated, in
accordance with step 762 using the sensing unit 100, the quarter
wave retarder plate 712 and the curved mirror 708 to thereby
produce a larger polarization that is more readily detectable. In
that regard, at step 766, a sufficient number of partial light
transmissions is generated by which some light from the reflected
light on the curved mirror 708 passes therethrough. At step 770,
such partial light transmissions are collected using the focusing
lens/analyzer plate 716. The numerous partial light transmissions
are then detected by the detector unit 720 at step 774. The
detector unit 720 uses the intensities of the polarized light to
generate a light signal that relates to the mass of the sensing
unit 100. At step 778, the value of this sum light signal is
compared with the previously determined standard data as part of
determining whether or not the analyte of interest is present.
Test Piece Containing Device
[0120] In one embodiment, with reference to FIGS. 23 and 24, the
system also includes device 800 for containing a test piece having
a number of sensing units 100 to be tested. Unlike known prior art,
the device 800 includes a combination of assemblies and elements
that are used, either alone or together, to precisely control
heating, humidity, cross-contamination and mixing of materials with
or part of sensing units 100. The device 800 includes an enclosure
unit 804 (FIG. 15) and a base plate 808 for housing a number of
assemblies useful in providing such functions. An insulating cover
806 is preferably provided over the enclosure unit 804 in order to
reduce the amount of heat loss to the environment outside of the
device 800, which effectively reduces the convection inside of the
device 800. When convection is reduced, there is less unwanted
evaporation. An inner lid 810 is also provided with the insulating
cover 806. The inner lid 810 geometrically influences the
convection pattern created by a temperature gradient caused by the
heating assembly 812 (higher temperature) and the walls (lower
temperature) of the device 800. The inner lid 810 is curved in way
that prevents condensation from dripping down onto the sensing
units and test piece. The base plate 808 has a sealing ring 814
(FIG. 24) located around its edge which forms a seal between the
base plate 808 and the insulating cover 806. This reduces the
amount of heat and humidity loss from the device 800 where the base
plate 808 and the cover 806 come into contact.
[0121] The heating assembly 812 is used to heat the sensing units
on the test piece 332 to a desired or predetermined temperature,
such as 37 degrees Celsius and maintain that temperature to within
at least .+-.1 degree Celsius. The heating assembly 812 includes a
plate sub-assembly 816 that is comprised of a heating element
situated between two stainless steel plates. These two plates act
as heat reservoirs and are essentially in direct contact with the
test piece and, by this arrangement, the heating assembly 812
provides heat to the sensing units by conduction. In order to
generate the heat, the plate sub-assembly 816 is powered by a
heater controller (not shown) that is electrically connected to the
plate sub-assembly 816 by a power cord 818 (FIG. 23). The heating
assembly 812 also includes a support frame 820 that surrounds at
least some of the periphery of the plate sub-assembly 816. The
support frame 820 is connected to lower hinge members 824a, 824b at
one end of the support frame 820. The lower hinge members 824a,
824b enable the heating assembly 812 to be pivoted for desired
access to the space below the heating assembly 812, as will be
understood from a subsequent description of the humidifier assembly
of the device 800. Disposed upwardly of the heating assembly 812 is
a barrier manifold 830 that is an elongated member having a number
of barrier slits 834 that are perpendicular to the elongated plane
of the barrier manifold 830. The barrier slits 834 have a
sufficient depth to receive barrier members 838 so that portions of
the barrier members 838 are held in the barrier slits 834 and other
portions of the barrier members 838 extend outwardly from the plane
of the barrier member 830. One such barrier member 838 is shown in
FIG. 23. The barrier members 838 are spaced a predetermined
distance from each other and such spacing is based on the spacing
between sensing units on a test piece. That is, the barrier
manifold 830 overlies the test piece and each of the sensing units
is separated from each of the other sensing units by one or more
barrier members 838. In one embodiment, the barrier members 838
include O-rings which contact the body of the test piece without
contacting the area where the test is to be taken. Hence, the
barrier manifold 830 with barrier members 838 prevents
cross-contamination of the sensing units 100. The barrier manifold
830 also allows the sensing units to the washed and dried while in
the device 800. The barrier manifold 830 is connected at its ends
to upper hinge members 842a, 842b. At their opposite end portions,
the upper hinge members 842a, 842b are connected to manifold arms
850a, 850b. This arrangement enables the barrier manifold 830 to be
pivoted relative to the plate sub-assembly 816. When a test piece
is to be placed in the device 800, the barrier manifold 830 is
pivoted away from the plate sub-assembly 816 and the test piece is
able to be placed on the plate sub-assembly 816. Then, the barrier
manifold 830 is pivoted downwardly over the test piece 332 and
provides a slight clamping pressure using the barrier members 838
in order to safeguard against cross-contamination.
[0122] The device 800 further includes a humidifier assembly 860
that is comprised of a number of absorbative members for
maintaining a relative humidity of 100% within the enclosure unit
804 and the base plate 808 in order to avoid unwanted loss of
sensing unit materials due to evaporation. The absorbative members
include a main absorbative member 864 that is positioned in a
cavity 868 of a base member 872 that is supported on the base plate
808. The main absorbative member 864 has a length that extends for
at least a substantial portion of the length of the plate
subassembly 816 and is positioned essentially directly below this
sub-assembly. The main absorbative member 864 is soaked with water
prior to its placement in the cavity 868. Two side absorbative
members 876a, 876b are also utilized and are located to the side of
the main absorbative member 864 adjacent to and on top of a number
of wells 880 formed in a wall 884 of the base member 872. The side
sponge members 876a, 876b are also soaked with water before placing
them in the device 800 next to the wells 880. During use, the side
sponge members 876a, 876b draw water from the wells 880 by means of
a wicking operation to maintain their soaked or moisture-laden
state in order to continue to provide the desired humidity.
[0123] The device 800 also includes a mixing assembly 892
(diagrammatically illustrated in FIGS. 23 and 24) which is provided
to enhance the mixing of reagents associated with the sensing units
100 to be analyzed. In that regard, and with reference to a single
drop of material(s) on the sensing unit 100, the motion and pattern
of diffusion has been observed to be a slow descending spiral that
varies with the location and orientation of the mixing assembly
892. When this motion within a drop is added to the motion provided
by convection due to the drop being heated, there is greater fluid
movement and mixing within each drop. In the preferred embodiment,
the mixing assembly 892 includes a mechanism for providing
oscillatory motion using an offset weight rotating about a fixed
axis. This rotation is accomplished by a miniature variable-speed
motor. Significantly, as illustrated in FIG. 23, the mixing
assembly 892, including the motor and the offset weight, is
situated in a compound axis orientation next to the base member 872
along its length. Preferably, the orientation angle is greater than
about 10 degrees but less than about 70 degrees relative to the
plane of the base plate 808.
[0124] In view of the foregoing description, a system is described
that includes a number of sub-system components that cooperate with
each other in the detection and/or measurement of an analyte of
interest using mass change. The system components include a test
piece on which a number of sensing units are located that are to be
tested in connection with determining whether one or more of them
has an analyte of interest. The test piece is dimensioned to be
held in a test piece containing device that provides a number of
functions in the preparation and care of the test piece with
sensing units for subsequent testing. Such functions include
precisely controlling the heating, humidifying, avoidance of
cross-contamination and mixing of materials that are with or part
of the sensing units. The sensing units include an attachment layer
that is able to resist delamination of the different layers. The
sensing unit may include an attachment layer that is used to
immobilize a ligand layer that is receptive to the analyte of
interest. The sensing unit may include a tripartite and/or dual
element attachment layer that is characterized by an insulating
layer located between an upper surface and a lower surface. The
insulating layer acts to prevent the transfer of unwanted effects
between the lower and upper binding surfaces. The sensing unit may
include the dual element attachment layer in which the lower
element has an organofunctional silial compound. Mass enhancement
systems are preferably utilized that can include a variety of
materials such as kinetic-active mass enhancement with one or more
desired enzymes associated with the substrate, passive mass
enhancement systems in which an existing mass/constant is used
and/or a self-assembling amplification system. A further system
component is the instrument that is utilized in determining whether
or not the analyte of interest is present on a particular sensing
unit. The instrument is highly sensitive and able to accurately
determine whether an analyte is present. Such an instrument may
include components for generating and analyzing multiple
reflections from the same sensing unit to enhance or amplify the
detected signal. The instrument may include optical elements by
means of which polarized light having only one component is
generated to enhance the detected signal that is indicative of the
presence of the analyte of interest.
[0125] The foregoing discussion of the invention has been presented
for purposes of illustration and description. Further, the
description is not intended to limit the invention to the form
disclosed herein. Consequently, variations and modifications
commensurate with the above teachings, within the skill and
knowledge of the relevant art, are within the scope of the present
invention. The embodiments discussed hereinabove are further
intended to explain the best mode known of practicing the
inventions and to enable others skilled in the art to utilize the
inventions in such, or in other embodiments and with the various
modifications required by their particular application or uses of
the inventions. It is intended that the appended claims be
construed to include alternative embodiments to the extent
permitted by the prior art.
* * * * *