U.S. patent application number 11/423669 was filed with the patent office on 2010-10-14 for tip tray assembly for optical sensors.
This patent application is currently assigned to ForteBio, Inc.. Invention is credited to Sae Choo, Scott Lockard, Michael W. Recknor, Hong Tan, Krista Leah Witte, Robert Zuk.
Application Number | 20100261288 11/423669 |
Document ID | / |
Family ID | 37571058 |
Filed Date | 2010-10-14 |
United States Patent
Application |
20100261288 |
Kind Code |
A1 |
Recknor; Michael W. ; et
al. |
October 14, 2010 |
TIP TRAY ASSEMBLY FOR OPTICAL SENSORS
Abstract
An apparatus and method for packaging of an optical sensing
fiber is disclosed. The apparatus includes a substrate with a
plurality of openings, and each opening is configured for holding
an optical sensing assembly. The assembly is positioned in the
opening with a tip of the assembly extending through the opening to
be suspended from the substrate. In addition, openings are arranged
so the assembly positioned therein avoids contacting another
assembly positioned therein. The apparatus can include a support
member for supporting the substrate and positioning the substrate
so the tip of the assembly suspended from the opening in the
substrate contacts solution in one of a plurality of wells in a
container adjacent to the substrate. The assembly can be configured
for preparing of the optical assembly for assay. An agitation
assembly for agitating the container to create flow of the solution
in the container wells over an optical sensing assembly is also
disclosed.
Inventors: |
Recknor; Michael W.;
(Oakland, CA) ; Tan; Hong; (San Jose, CA) ;
Zuk; Robert; (Atherton, CA) ; Witte; Krista Leah;
(Hayward, CA) ; Choo; Sae; (Sunnyvale, CA)
; Lockard; Scott; (Los Gatos, CA) |
Correspondence
Address: |
FENWICK & WEST LLP
SILICON VALLEY CENTER, 801 CALIFORNIA STREET
MOUNTAIN VIEW
CA
94041
US
|
Assignee: |
ForteBio, Inc.
Menlo Park
CA
|
Family ID: |
37571058 |
Appl. No.: |
11/423669 |
Filed: |
June 12, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
60690325 |
Jun 13, 2005 |
|
|
|
Current U.S.
Class: |
436/164 ;
356/427; 356/450; 422/69 |
Current CPC
Class: |
G01N 21/45 20130101 |
Class at
Publication: |
436/164 ;
356/427; 422/69; 356/450 |
International
Class: |
G01N 21/75 20060101
G01N021/75; G01N 21/84 20060101 G01N021/84; G01N 30/00 20060101
G01N030/00; G01B 9/02 20060101 G01B009/02 |
Claims
1.-34. (canceled)
35. An apparatus for maintaining flow of the solution, the
apparatus comprising an agitation assembly operably coupled to a
container with a plurality of wells containing solution, each well
being configured for holding a discrete optical sensing assembly
therein, the optical sensing assembly being configured for
measuring a characteristic of the solution wherein said measuring a
characteristic comprises kinetic analysis and wherein the agitation
assembly comprises an agitation device that moves the container
according to a specified type of motion to agitate the solution
relative to the optical assembly to create flow of the solution
relative to the optical assembly said flow being sufficient for
said kinetic analysis.
36. The apparatus of claim 35, wherein the agitation device is
driven by an electrical motor.
37. The apparatus of claim 35, wherein the specified type of motion
is repetitive motion of the solution in the well, relative to the
optical assembly.
38. The apparatus of claim 37, wherein the repetitive motion is
circular motion.
39. The apparatus of claim 35, wherein the agitation device is
driven by an actuator for creating repetitive motion that is not in
orbital trajectory.
40. The apparatus of claim 35, wherein the specified type of motion
is random motion of the solution in the well, relative to the
optical assembly.
41. The apparatus of claim 35, wherein the agitation device is
operably coupled with the optical assembly to move the assembly
relative to the sample.
42. The apparatus of claim 35, wherein the apparatus rests on a
surface suspended above the agitation device with suspensions, the
agitation device providing agitation of the container creating
surface flow of the solution relative to the optical assembly.
43. The apparatus of claim 35, wherein the agitation assembly
further comprises a heater adapter for providing heated flow of
solution.
44. The apparatus of claim 43, wherein the heater adapter is
positioned between the container and the agitation assembly and is
operable coupled to the container and the agitation assembly.
45. The apparatus of claim 35, wherein the agitation assembly is
included in a robotic instrument for assay with the container
mounted on the agitation assembly, the container and agitation
assembly being mounted in the instrument adjacent to a substrate
for holding an optical sensing assembly.
46. The apparatus of claim 45, wherein a robotic arm moves the
optical sensing assembly from the substrate to the container to dip
the assembly in the solution, the solution being simultaneously
agitated by the agitation device.
47. The apparatus of claim 35 wherein the kinetic analysis
comprises molecular binding kinetic analysis.
48. A method for measuring a characteristic of a solution, the
method comprising: providing the apparatus of claim 35; agitating
the solution; contacting the solution with the optical sensing
assembly; and measuring the characteristic of the solution with the
optical assembly wherein the measuring comprises kinetic
analysis.
49. The method of claim 48 wherein said measuring comprises
biolayer interferometry.
50. The method of claim 48 wherein the kinetic analysis comprises
molecular binding kinetic analysis.
51. The method of claim 48 wherein the characteristic comprises
molecular binding.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/690,325, filed on Jun. 13, 2005, entitled "Tip
Tray Assembly for Optical Sensors," the entire disclosure of which
is hereby incorporated by reference herein, including any
appendices or attachments thereof, in its entirety for all
purposes.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to an apparatus and method
based on fiber optic interferometry, and in particular, to a tip
tray apparatus for packaging of optical sensors used in detecting
analytes and mechanisms for creating flow of solution.
[0004] 2. Description of the Related Art
[0005] Diagnostic tests based on a binding event between members of
an analyte-anti-analyte binding pair are widely used in medical,
veterinary, agricultural and research applications. Typically, such
methods are employed to detect the presence or amount of an analyte
in a sample, and/or the rate of binding of the analyte to the
anti-analyte. Typical analyte-anti-analyte pairs include
complementary strands of nucleic acids, antigen-antibody pairs, and
receptor-receptor binding agent, where the analyte can be either
member of the pair, and the anti-analyte molecule, the opposite
member.
[0006] Diagnostics methods of this type often employ a solid
surface having immobilized anti-analyte molecules to which sample
analyte molecules will bind specifically and with high affinity at
a defined detection zone. In this type of assay, known as a
solid-phase assay, the solid surface is exposed to the sample under
conditions that promote analyte binding to immobilized anti-analyte
molecules. The binding event can be detected directly, e.g., by a
change in the mass, reflectivity, thickness, color or other
characteristic indicative of a binding event. Where the analyte is
pre-labeled, e.g., with a chromophore, or fluorescent or
radiolabel, the binding event is detectable by the presence and/or
amount of detectable label at the detection zone. Alternatively,
the analyte can be labeled after it is bound at the detection zone,
e.g., with a secondary, fluorescent-labeled anti-analyte
antibody.
[0007] Co-owned U.S. Pat. No. 5,804,453, (the '453 patent) which is
incorporated herein by reference, discloses a fiber-optic
interferometer assay device designed to detect analyte binding to a
fiber-optic end surface. Analyte detection is based on a change in
the thickness at the end surface of the optical fiber resulting
from the binding of analyte molecules to the surface, with greater
amount of analyte producing a greater thickness-related change in
the interference signal. The change in interference signal is due
to a phase shift between light reflected from the end of the fiber
and from the binding layer carried on the fiber end, as illustrated
particularly in FIGS. 7a and 7b of the '453 patent. The device is
simple to operate and provides a rapid assay method for analyte
detection.
[0008] The optical tip tray device described herein can be used
with a fiber-optic inferometer assay device, as described above.
Specifically it provides a mechanism for packaging and holding
discrete fiber optic sensors in a format that allows for easy use
of the sensors. Before the types of assays described above are
conducted, the sensors can undergo some type of pre-wetting.
Techniques can also be used to immobilize molecules, such as
proteins, to the surface of the sensor. "Pre-wet," as used herein,
is a procedure in which a sensor coated with immobilized binding
proteins is hydrated to restore their biological activity. Sensors
coated with proteins can be stored dry in order to preserve the
activity of the proteins until they are to be used in an assay. In
immobilization procedures, sensors are put into contact with sample
solutions, such as protein-containing samples, and the proteins or
other molecules in the sample are immobilized to the surface of
biosensors coated with the appropriate surface chemistry. In the
device disclosed herein, discrete optical sensors are packaged in a
format (e.g., a format that corresponds to the 96-well format of a
standard microtiter plate) that allows the sensors to be easily
dipped into pre-wet or protein-immobilization solutions. Thus, in
some embodiments, the device provides for off-line incubation,
pre-wet, and/or immobilization. In contrast, current devices for
holding biosensors are either in flow cell format or have sensors
located as a part of the bottom of a microplate well, both of which
require different pre-wetting and immobilization procedures. In
addition, these systems do not provide flexibility for users to
arrange or configure the biosensors to customize the sensor
arrangement for immobilization. Users do not have the option to
simply remove and save unused biosensors, but are instead forced to
use an entire set of sensors for each experiment even if only a few
were needed.
[0009] Therefore, there is a need for an easy mechanism for
off-line incubation, pre-wetting, and immobilization where the user
has the flexibility to move around the sensors and customize the
arrangement. There is also a need for a device that stores these
types of discrete sensors in a format for easy pick up of the
sensors, for transfer of the sensors to a second microplate for
assay, and for mapping of sensors to sample wells. Furthermore,
these types of discrete sensors need to be packaged to avoid damage
during shipping, handling, and storage of the sensors.
[0010] Current devices also have limitations with regard the
mechanism for providing flow in wells during an assay. For
molecular kinetic analyses and other types of analyses, the device
must include some sort of mechanism for providing flow (e.g.,
within the wells of the microtiter plate containing sample or the
second microtiter plate), for example, to measure the
disassociation of molecules from the sensor surfaces. Where the
sensor surface is located at the bottom of a microtiter plate,
however, it is difficult to create any flow. Without proper flow,
it is impossible to provide a valid environment for molecular
binding kinetic analysis. Current systems use flow cells to create
sample or buffer flow over the sensing surface. For example, some
current systems use microfluidics and fluidic channels to move the
fluid around to bring reagents into contact with a particular
biosensor. However, these types of designs put a large design and
support burden on the instrumentation. Thus, there is needed a
mechanism that allows for fluidic motion without the need for
microfluidics or fluidic channels. There is a need for a mechanism
that allows exposure of the biosensor to a relatively large bulk of
reagents by providing continuous flow of reagent over the
biosensor.
[0011] The present invention is designed to overcome these and
other limitations with a design that allows flexibility for
arrangement or configuration of biosensors, biosensor mapping
capability to sample wells in a microtiter plate, off-line
incubation, pre-wet, and immobilization, and an effective mechanism
for orbital flow of the reagent over the biosensors, among other
advantages.
SUMMARY OF THE INVENTION
[0012] The invention includes, in one aspect, an apparatus and
method for packaging of an optical sensing fiber. The apparatus
includes a substrate with a plurality of openings, and each opening
is configured for holding an optical sensing assembly. The assembly
is positioned in the opening with a tip of the assembly extending
through the opening to be suspended from the substrate. In
addition, openings are arranged so the assembly positioned therein
avoids contacting another assembly positioned therein. In some
embodiments, the apparatus further includes a support member for
supporting the substrate and positioning the substrate so the tip
of the assembly suspended from the opening in the substrate
contacts solution in one of a plurality of wells in a container
adjacent to the substrate. The assembly can be configured for
preparing of the optical assembly for assay.
[0013] In one particular design, the apparatus includes both a
cover and a base that can lock together around the substrate to
form an enclosure around the optical sensing assembly. A container
(e.g., a microtiter plate) can be positioned inside the base,
beneath the substrate, in a manner that allows the tips of the
optical assemblies to line up in a predetermined orientation for
immersion in the solution inside the wells of the container. The
discrete optical assemblies can be moved around and arranged within
the substrate to customize an array of assemblies. In some
embodiments, the wells in the container are filled with different
types of protein solution or another type of solution to customize
the array. In some embodiments, different sensors (or sensors
coated with different reagents) are arranged within the substrate
to customize the array. The optical assemblies can be mapped to
wells in a sample container or microtiter plate to allow a user to
keep track of the samples being assayed and the different sensors
being used in the assay.
[0014] In another aspect, the invention includes a method for
packaging an optical sensing assembly for assay. The method
includes placing a discrete optical sensing assembly in one of a
plurality of openings in a substrate that is supported by a support
member. The optical sensing assembly is positioned in the opening
with a tip of the optical sensing assembly extending through the
opening to be suspended from the opening in the substrate. In
addition, the openings are arranged so the optical sensing assembly
positioned therein avoids contacting another optical sensing
assembly positioned therein. In some embodiments, the method
further includes positioning the substrate so the tip of the
optical sensing assembly suspended from the opening in the
substrate contacts solution in one of a plurality of wells in a
container adjacent to the substrate. In addition, the method can
include preparing the optical sensing assembly for assay, such as
by immobilization or pre-wet of the optical assembly before
assay.
[0015] In one design, the substrate can hold the array of optical
assemblies on a robotic instrument. In this embodiment, the
substrate may or may not be positioned over a container of wells
(e.g., a microtiter plate containing protein samples for
immobilization on the sensor tips). One or more of the optical
assemblies can be moved by a robotic arm from the tip tray
apparatus to another location (e.g., a second microtiter plate
containing samples and mounted on the robotic instrument next to
the substrate). In some embodiments, the tip tray apparatus holds
the sensors in position for manual transfer by a standard pipette
or other device to another location (e.g., to a wastebasket or
other location). Still other embodiments of the method include
covering the substrate with a cover and setting the substrate over
the container that is resting in a base. The base and cover can
lock together to surround the optical sensing assembly for
storage.
[0016] In yet another aspect, the invention includes an apparatus
for maintaining flow of the solution. The apparatus includes an
agitation assembly operably coupled to a container (e.g., the
second sample-containing microtiter plate described above) with a
plurality of wells containing solution. Each of the wells is
configured for having a discrete optical sensing assembly immersed
therein. The optical sensing assembly is also configured for
measuring a characteristic of the solution. In addition, the
agitation assembly comprises an agitation device that moves the
container according to a specified type of motion to agitate the
solution relative to the optical assembly to create flow of the
solution relative to the optical assembly.
[0017] In one design, the agitation device can be a motor or an
actuator, or other type of device for providing movement of the
solution to create surface flow. The specified type of motion
created by the agitation device can be a repetitive or a random
motion of the solution, thus causing it to flow over the optical
sensing assembly for the assay. In some embodiments, the apparatus
is placed on a surface above the agitation device, and the device
thereby causes movement or vibration of the solution relative to
the optical sensing assembly.
[0018] In some embodiments of the invention, the substrate that
holds an array of sensors (e.g., as part of the tip tray apparatus)
over a first container (e.g., a first microtiter plate) is mounted
on a robotic instrument adjacent to a sample container (e.g., a
second microtiter plate containing sample) that is mounted on the
agitation assembly within the robotic instrument. A robotic arm
(e.g., a robotic system with 8 SMA's) can pick up one or more
sensors (e.g., a row of eight sensors) and transfer them over to
the second container outside the tip tray apparatus for dipping of
the sensors in the sample contained in the second container.
[0019] These and other objects and features of the present
invention will become more fully apparent when the following
detailed description of the invention is read in conjunction with
the accompanying drawings.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0020] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, and accompanying drawings, where:
[0021] FIG. 1 shows the basic system setup for the biosensor and
tip tray apparatus, according to an embodiment of the
invention;
[0022] FIG. 2 shows an interferometer system constructed according
to an embodiment of the invention;
[0023] FIGS. 3A and 3B show a portion of an interference wave over
7 peak and valley orders (3A), and over in a visible portion of the
spectrum (3B);
[0024] FIG. 4 shows an optical sensing assembly constructed
according to an embodiment of the invention;
[0025] FIG. 5 shows an apparatus including a substrate and an
optical sensing assembly stored in an opening in the substrate,
according to an embodiment of the invention;
[0026] FIG. 6 shows an exploded view of the apparatus illustrating
a cover, a substrate, a base, and a container of wells, according
to an embodiment of the invention;
[0027] FIG. 7 shows perspective view of the substrate with an array
of openings therein, according to an embodiment of the
invention;
[0028] FIG. 8 shows an exploded view of the container of wells and
a base structure, according to an embodiment;
[0029] FIG. 9 shows a perspective view of the cover for the
apparatus, according to an embodiment of the invention;
[0030] FIG. 10 shows a side view of the apparatus, according to an
embodiment of the invention.
[0031] FIG. 11 shows a cross-section of a side view of the
apparatus with the tip lined up with the well, according to a
embodiment of the invention;
[0032] FIG. 12 shows a cross-section of a side view of the
apparatus with the tip of the optical sensing assembly inside the
well, according to an embodiment of the invention;
[0033] FIGS. 13a and b show an apparatus for agitation of solution
in a well relative to an optical sensing assembly, according to an
embodiment of the invention.
[0034] FIG. 13c shows a robotic instrument for assay, according to
an embodiment of the invention.
[0035] FIG. 14 shows a graphical illustration of binding curves
with and without flow, according to an embodiment of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0036] Terms used in the claims and specification are to be
construed in accordance with their usual meaning as understood by
one skilled in the art except and as defined as set forth below.
Numeric ranges recited in the claims and specification are to be
construed as including the limits bounding the recited ranges.
[0037] The term "in vivo" refers to processes that occur in a
living organism.
[0038] An "analyte-binding" molecule refers to any molecule capable
of participating in a specific binding reaction with an analyte
molecule. Examples include but are not limited to, e.g.,
antibody-antigen binding reactions, and nucleic acid hybridization
reactions.
[0039] A "specific binding reaction" refers to a binding reaction
that is saturable, usually reversible, and that can be competed
with an excess of one of the reactants. Specific binding reactions
are characterized by complementarity of shape, charge, and other
binding determinants as between the participants in the specific
binding reaction.
[0040] An "antibody" refers to an immunoglobulin molecule having
two heavy chains and two light chains prepared by any method known
in the art or later developed and includes polyclonal antibodies
such as those produced by inoculating a mammal such as a goat,
mouse, rabbit, etc. with an immunogen, as well as monoclonal
antibodies produced using the well-known Kohler Milstein hybridoma
fusion technique. The term includes antibodies produced using
genetic engineering methods such as those employing, e.g., SCID
mice reconstituted with human immunoglobulin genes, as well as
antibodies that have been humanized using art-known resurfacing
techniques.
[0041] An "antibody fragment" refers to a fragment of an antibody
molecule produced by chemical cleavage or genetic engineering
techniques, as well as to single chain variable fragments (SCFvs)
such as those produced using combinatorial genetic libraries and
phage display technologies. Antibody fragments used in accordance
with the present invention usually retain the ability to bind their
cognate antigen and so include variable sequences and antigen
combining sites.
[0042] A "small molecule" refers to an organic compound having a
molecular weight less than about 500 daltons. Small molecules are
useful starting materials for screening to identify drug lead
compounds that then can be optimized through traditional medicinal
chemistry, structure activity relationship studies to create new
drugs. Small molecule drug compounds have the benefit of usually
being orally bioavailable. Examples of small molecules include
compounds listed in the following databases: MDL/ACD
(http://www.mdli.com/), MDL/MDDR (http://www.mdli.com/), SPECS
(http://www.specs.net/), the China Natural Product Database (CNPD)
(http://www.neotrident.com/), and the compound sample database of
the National Center for Drug Screening
(http://www.screen.org.cn/).
[0043] Abbreviations used in this application include the
following: "ss" refers to single-stranded; "SNP" refers to single
nucleotide polymorphism; "PBS" refers to phosphate buffered saline
(0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M
sodium chloride, pH 7.4); "NHS" refers to N-hydroxysuccinimide;
"MW" refers to molecular weight; "Sulfo-SMCC" refers to
sulfosuccinimidyl
4-[N-maleimidomethyl]cyclohexane-1-carboxylate.
[0044] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
ADVANTAGES AND UTILITY
[0045] The advantages and utility of the invention are illustrated
by reference to the Figures and Examples as described in greater
detail below. These include an apparatus that holds and stores
discrete optical fiber sensors in a format that is useful for
off-line pre-wetting or protein immobilization, for transfer with a
robotic or other type of instrument, etc. In some embodiments, a
substrate holds the sensors in a format that corresponds to a
standard 96-well microplate (i.e., an 8.times.12 format with
approximately 9 mm spacing between the sensors). This positioning
allows a microplate or other container to be placed in the tip tray
apparatus under the sensors so that each tip of each sensor is
suspended over and can be immersed in a liquid in the microplate
wells. This design provides a means for immersing the end of the
sensor so that the tips of the sensors do not have to rub against
or otherwise contact the sides of the package or one another, thus
protecting them from contamination or damage.
[0046] Holding the sensors in this type of format can serve a
number of purposes. As one example, this format can be useful in
pre-wetting of the sensors. The sensors can be coated with
immobilized binding proteins and stored dry in order to preserve
the activity of the proteins. Then a container, such as a
microtiter plate, containing simple buffer or other solution can be
placed in the tip tray beneath the sensors to immerse the tips of
the sensors in the buffer. This immersion allows the sensors to
become hydrated, thus restoring their biological activity prior to
their transfer to the sample containing microplate (e.g., a second
microtiter plate outside the tip tray) for assay. As another
example, this format can be useful in immobilization of molecules,
such as proteins, to the surface of the sensors (or e.g., for
measurement of interaction with another protein already immobilized
on the optical sensing assembly). For example, protein-containing
samples can be dispensed into microplate wells and the microplate
can be placed in the tip tray under the tips of the sensors that
have the appropriate surface chemistry so that binding of the
protein to the sensor occurs. These samples may be, e.g., the same
for all wells or different in different wells for preparing a
homogenous or heterogenous set of sensors. Since protein
immobilization does not entail optical thickness measurements, the
immobilization procedure can be performed off-line or outside of
the use of the instrument for assay (i.e., in some embodiments the
sensors do not have to be on the robotic instrument during
immobilization).
[0047] Another advantage of the apparatus described herein is that
the biosensors are each discrete structures that do not have to be
connected to one another or connected to the apparatus that
supports them (e.g., they are not located at the bottom of
microplate wells, etc), so a user can move around the various
sensors or customize the arrangement as desired. Since the sensors
are discrete, the user can remove and save extra sensors that are
not needed for a smaller-sized assay, rather than wasting these
unused sensors as might occur in an apparatus where the sensors are
located at the bottom of microplate wells. In addition, as
described above, the tip tray apparatus can have a microtiter plate
or other container optionally placed inside the base of the tip
tray apparatus for pre-wet (pre-conditioning of tips) or off-line
immobilization of proteins, and different proteins can be used in
each well of the plate, thereby allowing for a customized array of
sensor tips.
[0048] A further advantage includes the ability to map the sensors
and their specific arrangement to the sample container (e.g., a
second microtiter plate outside of the tip tray) including wells
filled with sample. The position of each sensor can be mapped to
the corresponding position of the well of sample that the sensor
will contact during assay. Yet other advantages include the design
of the sensors that allow them to be positioned for automatic
pick-up and usage by a robotic instrument or by a standard pipette.
In embodiments in which the sensors are positioned in a 96-well
format, the sensors can then be easily transferred to a second
microplate for measurement. Furthermore, the overall apparatus is
configured to protect the sensors during shipping, handling, and
storage. The sensors rest neatly in separate openings in the
apparatus and, in some embodiments, the apparatus includes a
hard-sided bottom portion and a top portion that effectively
surround the sensors in a safe and protected environment.
[0049] A further advantage is provided in the mechanism for
creating orbital flow without the need for microfluidics and
fluidic channels. As described above, this is needed for providing
a valid environment for molecular binding kinetic analysis. In the
invention described herein, an orbital agitation device is provided
for creating relative motion between an optical fiber sensor
surface and the samples in a microtiter plate. In one embodiment,
the plate is rotated in a plane that is perpendicular to the fiber
sensors. An angle between the plate bottom and sensor fibers is
also possible to reduce the reflected light (i.e., background
noise).
[0050] FIG. 1 shows, in schematic view, an interferometer system 20
constructed in accordance with the invention. In its most basic
elements, the system 20 includes a light source 22, a biosensor or
optical sensing assembly 26 that functions as a sensing element or
detector tip, and a detector unit 28 for detecting interference
signals produced by interfering light waves reflected from the
optical sensing assembly 26.
[0051] Light from source 22 is directed onto the optical sensing
assembly 26, and reflected back to the detector through an optical
coupling assembly indicated by dashed lines at 30. In a preferred
embodiment, the coupling assembly includes a first fiber cable 32
extending from the light source 22 to the optical sensing assembly
26, and a second fiber cable 34 which carries reflected light from
the optical sensing assembly 26 to the detector 28. Optionally, an
optical coupler may be used to optically couple the fiber cables
32, 34 to the optical sensing assembly 26.
[0052] The light source 22 in the system 20 can be a white light
source, such as a light emitting diode (LED) which produces light
over a broad spectrum, e.g., 400 nm or less to 700 nm or greater,
typically over a spectral range of at least 100 nm. Alternatively,
the light source 22 can be a plurality of sources each having a
different characteristic wavelength, such as LEDs designed for
light emission at different selected wavelengths in the visible
light range. The same function can be achieved by a single light
source 22, e.g., white light source, with suitable filters for
directing light with different selected wavelengths onto the
optical sensing assembly 26.
[0053] The detector 28 can be a spectrometer, such as
charge-coupled device (CCD), capable of recording the spectrum of
the reflected interfering light from the optical sensing assembly
26. Alternatively, where the light source 22 operates to direct
different selected wavelengths onto the optical sensing assembly
26, the detector 28 can be a simple photodetector for recording
light intensity at each of the different irradiating wavelengths.
In still another embodiment, the detector 28 can include a
plurality of filters which allows detection of light intensity,
e.g., from a white-light source, at each of a plurality of selected
wavelengths of the interference reflectance wave.
[0054] FIG. 2 shows the optically functional part of an optical
sensing assembly 26 constructed in accordance with one embodiment
of the invention. The optical sensing assembly 26 includes a short
length of optical fiber 42, to which the remainder of the optical
sensing assembly 26 is fixedly attached. The other end of optical
fiber 42 couples to the fiber cables 32 and 34, respectively. As
seen, the assembly 26 includes a transparent optical element 38
having first and second reflecting surfaces 42, 40 formed on its
lower (distal) and upper (proximal) end faces, respectively.
According to an important feature of the invention, the thickness
"d" of the optical element 38 between its distal and proximal
surfaces, i.e., between the two reflecting surfaces, is at least 50
nm, and preferably at least 100 nm. An exemplary thickness is
between about 100-5,000 nm, preferably 400-1,000 nm. The first
reflecting surface 42 is formed of a layer of analyte-binding
molecules, such as molecules 44, which are effective to bind
analyte molecules 46 specifically and with high affinity. That is,
the analyte and anti-analyte molecules are opposite members of a
binding pair of the type described above, which can include,
without limitations, antigen-antibody pairs, complementary nucleic
acids, and receptor-binding agent pairs.
[0055] The index of refraction of the optical element 38 is
preferably similar to that of the first reflecting surface 42, so
that reflection from the lower distal end of the end optical
sensing assembly 26 occurs predominantly from the layer formed by
the analyte-binding molecules 44, rather than from the interface
between the optical element 38 and the analyte-binding molecules
44. Similarly, as analyte molecules 46 bind to the lower layer of
the optical sensing assembly 26, light reflection form the lower
end of the assembly 26 occurs predominantly from the layer formed
by the analyte-binding molecules 44 and bound analyte 46, rather
than from the interface region. One exemplary material forming the
optical element 38 is SiO.sub.2, e.g., a high-quality quality glass
having an index of refraction of about 1.4-1.5. The optical element
38 can also be formed of a transparent polymer, such as polystyrene
or polyethylene, having an index of refraction preferably in the
1.3-1.8 range.
[0056] The second reflecting surface 40 in the optical sensing
assembly 26 formed as a layer of transparent material having an
index of refraction that is substantially higher than that of the
optical element 38, such that this layer functions to reflect a
portion of the light directed onto the optical sensing assembly 26.
Preferably, the second layer has a refractive index greater than
1.8. One exemplary material for the second layer is Ta.sub.2O.sub.5
with refractive index equal to 2.1. The layer is typically formed
on the optical element 38 by a conventional vapor deposition
coating or layering process, to a layer thickness of less than 50
nm, typically between 5 and 30 nm.
[0057] The thickness of the first (analyte-binding) layer is
designed to optimize the overall sensitivity based on specific
hardware and optical components. Conventional immobilization
chemistries are used in chemically, e.g., covalently, attaching a
layer of analyte-binding molecules to the lower surface of the
optical element. For example, a variety of bifunctional reagents
containing a siloxane group for chemical attachment to SiO.sub.2,
and an hydroxyl, amine, carboxyl or other reaction group for
attachment of biological molecules, such as proteins (e.g.,
antigens, antibodies), or nucleic acids. It is also well known to
etch or otherwise treat glass a glass surface to increase the
density of hydroxyl groups by which analyte-binding molecules can
be bound. Where the optical element 38 is formed of a polymer, such
as polystyrene, a variety of methods are available for exposing
available chemically-active surface groups, such as amine,
hydroxyl, and carboxyl groups.
[0058] The analyte-binding layer 44 is preferably formed under
conditions in which the distal surface of the optical element 38 is
densely coated, so that binding of analyte molecules 46 to the
layer forces a change in the thickness of the layer, rather than
filling in the layer. The analyte-binding layer 44 can be either a
monolayer or a multi-layer matrix.
[0059] The measurement of the presence, concentration, and/or
binding rate of analyte 46 to the optical sensing assembly 26 is
enabled by the interference of reflected light beams from the two
reflecting surfaces 40, 42 in the optical sensing assembly 26.
Specifically, as analyte molecules 46 attach to or detach from the
surface, the average thickness of the first reflecting layer 42
changes accordingly. Because the thickness of all other layers
remains the same, the interference wave formed by the light waves
reflected from the two surfaces is phase shifted in accordance with
this thickness change.
[0060] Assume that there are two reflected beams: The first beam is
reflected from the first surface, which is the distal end interface
between analyte-binding molecules 44 and bound analyte 46 and the
surrounding medium; and the second beam is reflected from the
second surface, which is the proximal interface between the optical
element (the first layer) and the high-index of refraction layer
(the second layer). The overall wavelength-dependent intensity of
the interference wave is:
I = I 1 + I 2 + 2 I 1 I 2 cos ( 2 .pi..DELTA. .lamda. )
##EQU00001##
where I is the intensity, I.sub.1 and I.sub.2 are the intensity of
two interference beams, .DELTA. is the optical path difference, and
.lamda. is the wavelength.
[0061] When (2.pi..DELTA./.lamda.)=N.pi., the curve is at its peak
or valley if N is an integer 0, 1, 2, . . . . The thickness of the
first layer d=.DELTA./2n. Therefore, .lamda.=4nd/N at peaks or
valleys (extrema). For the first several values of N, i.e., 0, 1,
2, . . . 7, and assuming a d of 770 nm, the equation gives:
[0062] N=0: .lamda.=.infin. (peak)
[0063] N=1: .lamda.=4nd=4,496.80 nm (Valley)
[0064] N=2: .lamda.=2nd=2,248.40 nm (Peak)
[0065] N=3: .lamda.=4nd/3=1,498.9 nm (Valley)
[0066] N=4: .lamda.=nd=1,124.20 nm (Peak)
[0067] N=5: .lamda.=4nd/5=899.36 nm (Valley)
[0068] N=6: .lamda.=2nd/3=749.47 nm (Peak)
[0069] N=7: .lamda.=4nd/7=642 nm (Valley)
[0070] N=8: .lamda.=nd/2=562 nm (Peak)
[0071] N=9: .lamda.=4nd/9=499.64 nm (Valley)
[0072] N=10: .lamda.=4nd/10=449.6 nm (Peak)
As can be seen, and illustrated further in FIGS. 3A and 3B, at
least three peaks/valleys (N=7-9) occur in the visible spectral
range.
[0073] If the 7.sup.th order valley is used to calculate the change
in molecular layer thickness, when the molecular layer attached to
the first layer increases from 0 nm to 10 nm, the 7.sup.th order
valley will shift to 650.74 nm. Therefore, the ratio between the
actual the phase shift of the 7.sup.th order valley and thickness
change equals (650.74-642.40)/10=0.834.
[0074] By contrast, if the initial spacing between the two
reflecting layers 40, 42 is made up entirely of the analyte-binding
molecules 44 on the end of the fiber, assuming a thickness of this
layer of 25 nm, then the first order peak will occur at 146 nm,
clearly out of the range of the visible spectrum, so that the
device will only see a portion of the region between the 0-order
valley and the first order peak, but will not see any peaks, making
a shift in the spectral characteristics of the interference wave
difficult to measure accurately.
[0075] Not until the total thickness of the reflecting layer
approaches about 100 nm will the first-order peak appear in the
visible spectrum. Assuming a total thickness change of up to 50 nm,
the thickness of the optical element can then be as small as 50 nm,
but is preferably on the order of several hundred nm, so that the
phase shift or change in periodicity of the interference wave can
be measured readily by a shift in the spectral positions of
higher-order peaks or valleys, e.g., where N=3-10.
[0076] The ratio between the actual thickness and the measured
phase shift is considered as a key factor of measurement
sensitivity. It can be appreciated how one can adjust the thickness
of the optical element 38 and its refractive index to improve and
optimize the sensitivity to accommodate the electronics and optical
designs.
[0077] Referring now to FIG. 4, there is shown an illustration of a
side view of the full optical sensing assembly 26 (or biosensor or
sensor), according to an embodiment of the invention. The sensors
can be fabricated from cut lengths of optical fiber 42 bonded into
a hub 404, similar in structure to a hypodermic needle and its hub.
A portion of the fiber 42 can be immersed in sample during an
assay. As shown in FIG. 2, a portion of the fiber 42 can be coated
with a substance, such as analyte-binding molecules 44, to which
the sample (e.g., analyte molecules 46) will bind. In some
embodiments, the analyte-binding molecule is a protein, a small
molecule, a nucleic acid, a carbohydrate, or some other type of
molecule. Where the molecule is a protein, the protein can be an
avidin, a streptavidin, an antibody, an antibody fragment, or
another type of protein. In some embodiments, the optical sensing
assembly 26 is used to measure the amount of analyte in the sample
based on the amount of analyte that binds to the analyte-binding
molecule coated on the assembly 26. In other embodiments, the
optical sensing assembly 26 is used to measure the kinetics of a
binding reaction between the analyte in the sample and the
analyte-binding molecule coated on the optical sensing
assembly.
[0078] Before these types of assays are conducted, the optical
sensing assemblies 26 can be immersed in a specific
molecule-containing immobilization solution for immobilization of
molecules, such as proteins, to the assemblies 26 that are coated
with the appropriate surface chemistry for this immobilization.
Similarly, the assemblies 26 can be immersed into a pre-wet
solution to hydrate the sensors restoring biological activity of
previously bound molecules (e.g., binding proteins) just prior to
the transfer of the sensors to a second microplate or other
container for assay, as explained above.
[0079] The hub 404 of the optical sensing assembly 26 extends from
fiber 402 and provides a base onto which a robotic instrument,
standard pipette, or other instrument can attach to the sensor and
move it from a first location to a second location. Specifically,
the instrument used for moving the optical sensing assembly 26 can
be attached at opening 406, and the assembly 26 can thus be moved
to a different location as desired. The instrument for moving the
assembly 26 can be designed for moving an array of assemblies 26 at
one time, and thus the instrument can pick up a number of the
discrete assemblies 26 from the tip tray holding the assemblies 26
(see FIG. 5) to move the assemblies 26 to a second location (e.g.,
a second microtiter containing sample to be tested).
[0080] FIG. 5 shows a tip tray apparatus 500 for packaging one or
more optical assemblies 26, according to an embodiment of the
present invention. The tip tray apparatus 500 includes a substrate
504, which is the portion of the tip tray apparatus 500 that
includes a number of openings 508 for containing one or more
discrete optical assemblies 26. The optical sensing assemblies 26
are discrete in that they are not connected or attached directly to
other optical assemblies 26, and each assembly 26 can be moved
around within the tip tray 500 to any configuration the user
selects. Other devices may be attached to the optical sensing
assemblies 26 in the tray 500, such as a standard pipette device, a
robotic instrument, an optical fiber assembly (as shown in FIG. 1),
etc., and these devices may indirectly and temporarily link these
assemblies 26 together for a particular purpose. However, the user
can still have the ability to move the assemblies 26 around at some
point to configure them for the assay. In some embodiments, the
optical sensing assemblies 26 are disposable tips that are to be
used for an assay and then thrown away and replaced by new tips for
the next assay.
[0081] The optical assemblies 26 are suspended in the openings 508
with the tip of the optical sensing assembly 26 extending away from
the substrate 504. Each discrete optical sensing assembly 26 can
rest in an opening 508 in the substrate 504, and the optical
sensing assembly 26 is positioned in the opening so that the tip of
the optical sensing assembly 26 is suspended below the substrate
504 while the hub of the optical sensing assembly 26 rests above
the substrate 504. Thus, in FIG. 5, the hub 404 portion of the
optical sensing assembly 26 can be seen resting above the opening
508, while the fiber 42 portion of the assembly 26 is positioned
through the opening 508 and below the substrate 504. In some
embodiments, the optical sensing assembly 26 rests loosely within
the opening 508. In other embodiments, the optical sensing assembly
26 is snapped or locked into place in the opening 508. In some
embodiments, the substrate 504 includes an array of 96 openings 508
in an 8.times.12 pattern, similar to the structure of the wells in
a standard 96-well microtiter plate. In other embodiments, the
substrate 504 includes an array of 384 openings 508 or 1536
openings, similar to the wells in a standard 384-well or 1536-well
microtiter plate. However, the array of openings 508 can be
arranged in a number of other ways and can include any other number
of openings 508, as suitable. In general, the openings are arranged
so that the optical sensing assemblies 26 positioned in the
openings avoid contacting other optical sensing assemblies 26, and
thus the optical sensing assemblies 26 remain as discrete sensors
within the tip tray.
[0082] Also illustrated in FIG. 5 is a base structure 506 and a
cover 502 that make up a part of tip tray apparatus 500. The
substrate 504 rests on the base 506 and the cover 502 rests on top
of the base 506 to enclose the upper portion of the substrate 504.
In this manner, the optical assemblies 26 in the substrate 504 are
protected from the environment in the closed compartment created by
base 506 and cover 502. The optical assemblies 26 can be stored in
an enclosed manner for protection, and the assemblies 206 can also
be protected during shipping and handling. The hard-sided base 506
and cover 502 can lock into an enclosed position and thus protect
the contents of apparatus 500. The base 506 and cover 502 can form
an injection-molded plastic box, as shown in FIG. 5, or the
apparatus 500 can be constructed from materials other than plastic.
As shown in FIG. 5, the cover 502 can be transparent or
translucent, allowing the user to see the optical assemblies 26 and
substrate 504 beneath the cover 502.
[0083] Referring now to FIG. 6, there is shown an exploded view of
the tip tray apparatus 500, according to an embodiment of the
invention. This Figure illustrates how the various pieces of the
tip tray apparatus 500 fit together. As shown in FIG. 5, each of
the pieces of the apparatus can be completely separated from one
another. The cover 502 can be completely removed from the substrate
504 and base 506, to expose the optical assemblies 26 below.
Furthermore, the substrate 504 can be completely separated from the
base 506 to expose container 602 below. Container 602 can be a
standard 96-well, 384-well, or 1536-well microtiter plate that
includes an array of wells 604 for holding sample. Container 602
can also include any other number of wells (e.g., 1 well, 8 wells,
384 wells, etc.) and any other well arrangement or overall
configuration, as desired. Container 602 can be an injection-molded
plastic plate, as shown in FIG. 5, or container 602 can be made of
another material, as suitable.
[0084] As FIG. 6 illustrates, the openings 508 in the substrate 504
correspond generally with the wells 604 in container 602. When the
tip of an optical sensing assembly 26 is placed into an opening 508
in the substrate 504, the fiber 42 portion (not shown) of the
optical sensing assembly 26 will extend below the substrate 504 and
into the well 604 of container 602. The substrate 504 can be
arranged to be a distance from container 602 that permits a portion
of fiber 42 (not shown) of the optical sensing assembly 26 to be
immersed in sample contained in the well 604. The container 602 can
also be separated from the base 506 to allow the container to be
removed from the apparatus 500. Thus, a user can place pre-wet or
immobilization solution in the wells 604, for example, while the
container 602 is separate from the tip tray apparatus 500, and the
user can then insert the container 602 into the base 506 when the
wells 604 have been filled. In addition, the substrate 504 can be
used by itself without the container 602. For example, the
substrate 504 can be mounted on a robotic instrument and can simply
hold the optical assemblies in place for a robotic arm to attach to
one or more of the assemblies and move the assemblies to a
container of sample to be tested (e.g., a microtiter plate that is
separate from the tip tray apparatus). In this example, the
container 602 can be included under the substrate 504 on the
robotic instrument, but the container 602 can be optionally left
out, as desired.
[0085] The apparatus 500 described herein is useful in a number of
ways. As described previously, the apparatus 500 is useful in
orienting and positioning the optical assemblies 26 (i.e., in a
96-well format) so that they can easily be picked up by a robotic
instrument or a standard pipette and transferred to another
location. In addition, the apparatus 500 protects the optical
sensing assemblies 26 during shipping, handling, and storage by
enclosing them within a protected compartment. The apparatus 500
also provides a means to map selected sensors to selected wells in
a microtiter plate or other container. Furthermore, as explained
above, the apparatus can be used in incubation, immobilization,
pre-wet, etc. In some embodiments, the immobilization is conducted
off-line, separate from the robotic instrument used for assay.
However, in some embodiments, the substrate 504 plus container 602
filled with solution for immobilization of molecules to the optical
assemblies 26 (e.g., protein solution) can be placed onto the
robotic instrument. In some embodiments, the immobilization step
occurs while the substrate 504 is on the instrument.
[0086] In some embodiments, the apparatus 500 or some of the
components of the apparatus 500 can be mounted on a robotic system,
as described above. In some embodiments, the substrate 504 is
placed above a first microtiter plate containing samples or
solution (e.g., protein immobilization solution). The substrate 504
can be mounted alongside a second microtiter plate (that is
separate from the substrate 504) also mounted on the robotic
system. The second plate can contain the same or different samples
included in the first plate. One or more of the optical assemblies
26 in the substrate 504 can be picked up with a robotic system and
transferred to the second plate for immersion in the samples to be
tested. In some embodiments, the first microtiter plate is not
included, and instead only the substrate 504 is mounted on the
instrument. In some embodiments, the second plate is mounted on an
agitation assembly for creating orbital flow of sample within the
wells of the second plate, as will be described in more detail
below. In still other embodiments, the substrate 504 (or possibly a
second substrate 504) is used to position the sensors over the
second microtiter plate (or over another container holding samples
to be tested) during an assay.
[0087] Numerous different types of assays can be conducted using
the discrete optical assemblies 26. For example, assays can involve
an anti-species antibody carried on the sensor tip, for screening
hybridoma expression lines for cell lines with high antibody
expression, or an antigen carried on the tip, to characterize high
affinity antibodies against that antigen. Other assays can include
a protein carried on the tip, for identifying and characterizing
binding partners (DNA, RNA, proteins, carbohydrates, organic
molecules) for that protein, or a carbohydrate or glycosyl moiety
carried on the tip, for identifying and characterizing binding
partners (such as, e.g., DNA, RNA, proteins, carbohydrates, organic
molecules) for that carbohydrate. Still other assays can include a
protein thought to participate in a multi-protein complex carried
on the tip, for characterizing the binding components and/or
kinetics of complex formation, or a small protein-binding molecule
carried on the tip, for identifying and characterizing protein
binders for that molecule. These are but a few examples of assays
that could be conducted, and these are in no way meant to limit the
scope of the invention.
[0088] In some embodiments, every opening 508 in the substrate 504
contains an optical sensing assembly 26, and every well 604 in
container 602 contains a solution (i.e., an immobilization or
pre-wet solution, a sample, etc.) that is contacted by each optical
sensing assembly 26. However, the user can move around the
assemblies 26 and customize the array of assemblies 26, as desired.
For example, the user may wish to only use one row of optical
assemblies 26 for an assay, and may use only a corresponding row of
wells 604 in container 602. The user can leave all of the other
openings 508 in the substrate 504 and all of the other wells 604 in
the container 602 empty during pre-wetting and immobilization, for
example. In this manner, the user can avoid wasting a number of
sensors needlessly by simply omitting the sensors that are not
going to be used during an assay. In contrast, in an apparatus in
which the sensors are all included at the bottom of wells in a
microtiter plate, an entire set of sensors must be used for each
assay, even if the assay only includes 10 wells filled with sample.
In some embodiments, the user may wish to use different types of
samples or protein immobilization solutions in different wells 604
or different kinds of optical assemblies 26 (or assemblies 26 with
different coatings on the tip) to customize the array.
[0089] As explained above, the apparatus 500 arranges the optical
assemblies 26 in a manner that allows the optical assemblies 26 to
be mapped to the array of wells in the sample container or second
microtiter plate that will be used in conducting the assay. A
combination of software can provide programmable control of the
sample plate and of which samples are tested and/or which sensors
are used (e.g., different sensors coated with different proteins).
In some embodiments, the user can view the assay on a computer
display or other type of display, and thus can see which wells in
the sample plate are filled with which types of sample and/or can
keep track of which sensors are used and into which sample wells
these sensors are dipped into in the sample-containing second
microtiter plate. In this manner, the user will know what optical
assemblies 26 are associated with what samples.
[0090] Referring now to FIG. 7, there is shown an illustration of
the substrate 504 and openings 508, according to an embodiment of
the invention. The openings 508 of the substrate 504 extend down
below the substrate 504 to a sufficient distance to prevent the
optical assemblies 26 inside the openings 508 from tilting or
swaying from side to side a substantial amount. The depth of the
openings 508 will be illustrated more clearly in FIGS. 11 and
12.
[0091] In some embodiments, the substrate 504 includes one or more
support members 704 that extend from the substrate 504 and provide
support. In the embodiment of FIG. 7, the substrate 504 includes
four support members 704, one at each corner of the substrate.
However, these support members can be designed in various ways and
there can be more than four or fewer than four, as suitable. The
support members 704 provide support to the substrate 504, and can
support the substrate 504 when resting on a surface, on a robotic
instrument, in the base 506, or other locations. In some
embodiments, the substrate 504 is designed to be a sufficient
height that when the substrate 504 is loaded with optical
assemblies 26 and set on a surface without the base 506 or
container 602 below, the tips of the optical assemblies 26 will
still not contact the surface or contact each other. The support
members 704 allow the substrate 504 to be positioned on the base
506 by extending through slots in the base (see FIG. 8). The
support members 704 can slide into the openings in the base 506 and
the notches 706, 708 further contact the base to allow the
substrate 504 to be securely positioned on the base 506.
[0092] In some embodiments, the support members 704 are designed to
extend down into a robotic instrument platform or other device to
locate the sensors with respect to the instrument robotics. The
support members 704 can make contact with a mating surface in the
instrument and provide datum surfaces for location during
installation. Thus, any position tolerance increase that might have
been contributed to by the base 506 is therefore avoided, since the
base 506 does not need to be used for location in the
instrument.
[0093] The substrate 504 illustrated in the embodiment of FIG. 7
also includes a raised edge 702 that surrounds the center surface
of the substrate 504 and a recess 709 into which the optical
assemblies 26 are placed. Thus, the optical assemblies can be
positioned down into the recess 709 and can be protected by the
edge 702. The edge 702 provides the sensors with some protection
against getting knocked out of the openings 508 when the cover 502
is not in place. The recess 709 further provides some protection of
the sensors when the cover 502 is in place, since the cover has a
corresponding recess that helps to hold the sensors in position
(see FIG. 9).
[0094] FIG. 8 shows an exploded view of the base 506 and container
602, according to an embodiment of the invention. The container 602
can be placed in the base 506 so that the ends of the optical
assemblies 26 are aligned with the wells 604 in the container 602.
FIG. 8 also illustrates that container 602 can be removed from the
base 506 and replaced as required. Where the container 602 is a
standard microtiter plate, the used plate can easily be removed
after use in pre-wetting or immobilization and replaced with a new,
clean plate, as needed.
[0095] As described previously, the base 506 can include openings
or slots 802 for placement of the substrate 504. In the embodiment
of FIG. 8, there are four slots 802 in the base 506, one at each
corner. As the substrate 504 is placed over the base 506, the
substrate 504 is guided into position while preventing contact
between the ends of the optical assemblies 26 and other surfaces.
The support members 704 of the substrate 504 are guided into the
slots 802 in the base to provide secure positioning of the
substrate 504 in the base 506. The base 506 thus can protect the
tips of the optical assemblies 26 when the substrate 504 is
positioned on the base 506 without the container 602 in between.
The base 506 can also assist in guiding the substrate 504 into
place for the proper positioning of the optical assemblies 26 in
the wells 604 when the substrate 504 is positioned on the base 506
over container 602.
[0096] In some embodiments, the base 506 also includes a mounting
notch 804 that mates with the corner orientation notch 806 of the
container 602. In this manner, the user can easily find the correct
orientation of the container 602 in the base 506. The container 602
can be secured into position by sliding the orientation notch 806
under the mounting notch 804 to force the container 602 into its
specified orientation.
[0097] Referring now to FIG. 9, there is shown the cover 502 for
the apparatus 500, according to an embodiment of the invention. The
cover 502 can be generally contoured to fit the shape of the
substrate, including a raised outer edge 910 around the periphery
of the cover 502 and a recess 902 in the center of the cover 502.
The cover 502 fits over the top of the substrate 504 and protects
the hub 404 end of the optical sensing assembly 26 from
environmental contamination or other damage. The cover 502 can be
designed to fit such that the sensors cannot lift from the holes in
the substrate 504, regardless of the position of the apparatus 500
in space. For example, in this embodiment, even if the apparatus
500 is held upside down, the cover will retain the sensors in
position in the opening 508 of the substrate 504. One feature that
allows the cover 502 to hold the optical assemblies 26 firmly in
place is the recess 902 in the top of the cover. This recess 902
corresponds with the recess 709 in the substrate 504, and thus the
center portion of the cover 502 extends down into the recess 709 of
the substrate 504. In this manner, the center portion of the cover
502 is lowered close to the hubs 404 of the optical assemblies 26
when the assemblies 26 are positioned in the openings 508 of
substrate 504 to maintain the assemblies 26 in the openings
508.
[0098] In some embodiments, the cover includes structures 904 and
906 that engage the base 506 to snap the cover 502 into position on
the base 506. Various other types of locking mechanisms can be
used, as well, for locking the cover to the base. In some
embodiments, the cover 502 and the base 506 lock into the substrate
504 rather than locking into each other. In some embodiments, the
cover 502 further includes flap 908 that engages a notched portion
of the base 506 (that will be illustrated in FIG. 10), and provides
a gripping means for the user to grip the cover 502 and separate
the cover 502 from the apparatus 500. In addition, the recess 902
can also provide for nestled stacking of assemblies 500 during
storage. The base 506 includes feet (that will be illustrated in
FIG. 10) that will rest in the recess 902 when one apparatus is
placed on top of another, thus securing the stacked
configuration.
[0099] FIG. 10 shows a side view of the apparatus 500, according to
an embodiment of the invention. FIG. 10 further illustrates the
flap 908 of the cover 502, and how this flap 908 rests against
notch 1010 in the base, according to an embodiment of the
invention. The notch 1010 provides a space into which a user can
slip his/her fingers to grip the flap 908 and remove the cover 502.
In addition, FIG. 10 illustrates the stacking feet 1012 of base 506
that permit nestled stacking of assemblies 500, according to an
embodiment. In this embodiment, the feet 1012 of one apparatus 500
can rest in the recess 902 of another apparatus 500, thus holding
the two assemblies 500 in place during storage. The stacking feet
500 can also provide support for the apparatus 500 when the
apparatus is resting on a surface. FIG. 10 further illustrates the
support members 704 of substrate 504, and shows how these engage
the base for secure fitting, according to an embodiment of the
invention.
[0100] FIG. 11 shows a cross-sectional view of the side of the
apparatus 500, including the substrate 504, the container 602, and
the base 506, according to an embodiment of the invention. FIG. 11
illustrates how the hub 404 rests at the top of the substrate 504,
and the fiber 42 is suspended below the substrate 504 from the
opening 508, according to some embodiments. The optical sensing
assembly 26 is positioned above one of the wells 604, and is
positioned for insertion of the fiber 42 inside the well 604.
[0101] FIG. 12 further shows another cross-sectional view of the
side of the apparatus 500, including the substrate 504, the
container 602, and the base 506, according to an embodiment of the
invention. This view shows the fiber 42 inside of a well 604 that
can be filled with solution or sample. The optical sensing assembly
26 is positioned so that only a portion of the fiber is inside the
well, and thus only a portion will contact the solution (however,
this positioning can vary as needed). FIG. 12 further illustrates
the length of the opening 508 in this embodiment. The opening 508
at the upper surface of the substrate 504 can have an elongated
barrel below the surface that extends a distance beyond the surface
of the substrate 504. This design can provide additional support
for the optical sensing assembly 26 to avoid tipping or swaying of
the optical sensing assembly 26 and to keep the assembly 26 from
contacting other nearby assemblies 26 or other surfaces.
[0102] While the many of the embodiments shown herein include both
a cover 502 and a base 506, these elements are optional. In some
embodiments, either the cover 502 or base 506, or both, are
excluded from the apparatus 500. In addition, the shape of the
apparatus 500 can vary, as suitable.
[0103] Referring now to FIGS. 13a and 13b, there is shown
illustrations of an orbital flow apparatus 1300, according to an
embodiment of the present invention. The orbital flow apparatus
1300 can include a container 1302, an agitation assembly 1306, an
agitation device 1306 and a heater adapter 1304. However, one or
more of these parts can be left out of the apparatus or other parts
can be added, as suitable for a given application. Instead of using
a flow cell or microfluidics to create surface flow in a well, the
invention disclosed herein describes a different manner for
creating flow. The orbital flow apparatus 1300 shown in FIG. 13
provides a mechanism for creating the flow needed in a sample well
of a container 1302 for a molecular binding kinetic analysis or
movement of buffer solution in a well. Without proper flow, it is
very difficult to measure the disassociation of molecules from the
sensor surfaces. Container 1302 is similar to container 602, and
thus it can be a 96-well microtiter plate as shown in FIGS. 13a and
b, a 384-well or 1536-well microtiter plate, or some other type of
container. The orbital flow mechanism allows for the exposure of a
biosensor or optical sensing assembly 26 to a relatively large bulk
of reagents. This apparatus 1300 can provide for continuous flow of
reagent over an optical sensing assembly 26 by rotating the
container 1302 to create orbital flow within the wells. FIG. 13b
illustrates an optical sensing assembly 26 as was shown in FIGS.
1-12. This assembly 26 is suspended in a solution in the wells of
container 1302. The apparatus 1300 is designed to create relative
flow between the assembly 26 and the sample in which the biosensor
is immersed by moving or rotating the container 1302 in a plane
perpendicular to the optical assemblies 26, according to a
specified type of motion (e.g., repetitive motion, random motion,
etc.).
[0104] One embodiment of the apparatus 1300 uses a repetitive flow
mechanism. In this embodiment, the mechanism creates a repetitive
motion, resulting in relative movement of the assembly 26 and the
sample in the well. This type of mechanism can be designed in a
number of manners. One example is shown in FIG. 13 that includes a
surface 1310, which can be a substantially flat surface that rests
on suspensions 1312. In the example shown in FIG. 13, there are
four flexible suspensions 1312, but any other type of mechanism can
be used. The example shown in FIG. 13 further includes an agitation
assembly 1306 with an agitation device 1308 (e.g., an electrical
motor or another type of device for providing movement or
agitation) that is positioned below the surface 1310 (or adjacent
to the surface 1310). The agitation device 1308 includes a driving
mechanism for the repetitive motion, in this embodiment. In the
example illustrated in FIG. 13, the motor rotates the container
1302 in a plane that is perpendicular to the optical sensing
assembly 26 to cause the agitation to occur. This movement of the
motor causes the sample to move relative to the assembly 26, thus
causing continuous flow of the sample over the biosensor surface.
In some embodiments, the orbital motion can be controlled from 10
rpm to 5000 rpm. In other embodiments, the motion can be controlled
from 1 rpm to 10,000 rpm, from 100 rpm to 2000 rpm.
[0105] Some embodiments of the apparatus 1300 include an actuator
(e.g. electrical motor, piezo actuator, solenoids, etc.) to create
a repetitive motion that is not in orbital trajectory. The possible
trajectories can include linear, elliptical, sinusoidal, etc., or
any combination of these motions in the three-dimensional
space.
[0106] Another embodiment of the apparatus 1300 uses a random flow
mechanism. In this embodiment, the mechanism generates a random
motion or vibration that agitates the sample in the wells, and thus
creates relative movement of sample against the biosensor surface
26. For example, an ultrasound source can be used to agitate the
sample or solution to create flow of the sample over the biosensor
26.
[0107] In still other embodiments, instead of causing orbital
movement of the solution inside the well relative to the optical
assembly 26, the assembly 26 is agitated while the solution is kept
substantially stable. An agitation assembly 1306 can be attached to
the assembly to cause the assembly 26 to move relative to the
sample, thus creating relative movement of the sample over the
biosensor. Additionally, both the solution and the optical assembly
26 can be agitated in some embodiments.
[0108] In addition, movement of either the optical assembly 26 or
the sample is not limited to circular or elliptical motion.
Agitation can be created using numerous other types of motion, such
as movement up and down (i.e., by moving the sample up and down or
the biosensor up and down), movement in a straight line,
vibrational movement, etc.
[0109] In some embodiments, the agitation assembly 1306 includes a
heater adapter 1304 that connects to the container 1302 and the
agitation assembly 1306 by being sandwiched in between the two. The
heater adapter 1304 is mounted onto the orbital flow device to
provide heated flow during binding measurements. FIG. 13 shows how
the pieces of apparatus 1300 fit together, and can lock into
place.
[0110] As described above, the orbital flow apparatus 1300 can be
included in a robotic instrument for assay. FIG. 13c better
illustrates this usage of the inventions described herein in a
robotic instrument for assay. Robotic instrument 1350 is shown in
FIG. 13c, and this instrument can be included as a part of
interferometer system 20, described above. In some embodiments,
container 1302 can be mounted to the agitation assembly 1306 and
can contain samples to be tested during assay (e.g., the second
microtiter plate referred to above). The container 1302 can be
mounted alongside substrate 504 that is holding optical assemblies
26. A robotic arm 1352 can transfer one or more of the assemblies
26 from the substrate 504 over to the container 1302 and can dip
the assemblies 26 into the samples in the wells of container 1302.
The agitation assembly 1306 causes agitation of the container 1302
while the tips of the assemblies 26 are dipped into the samples,
thus creating flow of the sample over the optical assemblies 26 for
kinetic analyses or other types of assays.
[0111] The following example illustrates a method of the invention
for creating orbital flow, but is in no way intended to limit its
scope.
EXAMPLES
[0112] Below is an example of a specific embodiment for carrying
out the present invention. The example is offered for illustrative
purposes only, and is not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for.
[0113] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology, within
the skill of the art. Such techniques are explained fully in the
literature. See, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences,
18th Edition (Easton, Pa.: Mack Publishing Company, 1990); Carey
and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B (1992).
Example 1
Multi-Concentration Kinetic Experiment with Direct Comparison of
Flow=0 and Flow=140 Rpm
[0114] This example demonstrates the creation of orbital flow of a
sample relative to the biosensor. In this example, a set of
biosensors was coated with an antibody against mouse IgG2b. The
analyte (mouse IgG2b) was added into an assay buffer (1 mg/mL
bovine serum albumin in phosphate buffered saline, 0.02% Tween-20)
at the concentration specified in FIG. 14. FIG. 14 shows, in two
graphs, the binding curves for the mouse IgG2b to the Anti-mouse
IgG2b-coated sensors where flow is equal to 0 (e.g., no flow;
depicted in the upper graph) and where flow is equal to 7 mm/sec
(depicted in the lower graph). Binding of the analyte to the
antibody was monitored using the technique of Biolayer
Interferometry, and binding was recorded as a nanometer (nm)
increase in thickness of the binding layer (i.e., the layer formed
by binding of the analyte to the anti-analyte) as a function of
time (in seconds). The graphs show that the orbital flow (i.e.,
where flow=7) eliminates the early "lag" phase of binding of a 2.5
ug/ml sample of mouse IgG2b to the coated sensor due to diffusional
barriers (i.e., which can be seen where flow=0). Therefore, the
apparatus 1300 for creating orbital flow allows for flow of the
sample over the biosensor resulting in binding without the "lag"
phase that occurs when the apparatus 1300 is not used to create
orbital flow.
[0115] While the invention has been particularly shown and
described with reference to a preferred embodiment and various
alternate embodiments, it will be understood by persons skilled in
the relevant art that various changes in form and details can be
made therein without departing from the spirit and scope of the
invention.
[0116] All references, issued patents and patent applications cited
within the body of the instant specification are hereby
incorporated by reference in their entirety, for all purposes.
* * * * *
References