Note: Descriptions are shown in the official language in which they were submitted.
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BEAD-BASED ANALYSIS OF A SAMPLE
Cross-reference to Related Applications
This application claims priority from U.S. patent application 16/845,458,
filed on April 10, 2020,
which is a continuation-in-part of U.S. patent application 16/368,707, filed
on March 28, 2019.
Background
This description relates to bead-based analysis of a sample.
To obtain all the useful information in a sample of whole blood of a patient
for purposes of
diagnosis, for example, requires not only a complete blood count (CBC) of the
various types of
blood cells in the blood sample and their hemoglobin content but also a
chemical analysis of
other components in the acellular portion of blood (e.g., the plasma). Such
other components can
include molecules and ions of various kinds.
Traditionally, both a CBC and a chemical analysis of blood are performed in a
lab on large
expensive machines using tubes of venous blood obtained by phlebotomy. Hours
or days may be
required for the chemical analysis to be completed and the results returned.
Summary
In general, an aspect of the disclosure is a method including attaching two or
more beads to each
unit of one or more units of a chemical component in a sample, to form, for
each unit of the
chemical component, a multi-bead complex including two or more beads and the
unit of the
chemical component; placing the sample on a surface of an image sensor; at the
image sensor,
receiving light originating at a light source, the received light including
light reflected by,
refracted by, or transmitted through the beads of the multi-bead complexes; at
the image sensor,
capturing one or more images of the sample from the received light; and
identifying, in at least
one of the images of the sample, separate multi-bead complexes, the
identifying of the separate
multi-bead complexes including associating the two or more beads of each of
the multi-bead
complexes based on proximity to one another.
Implementations may include one or a combination of two or more of the
following features. The
method includes identifying a presence of the chemical component based on the
identification of
the separate multi-bead complexes. The method includes identifying a level of
the chemical
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component based on the identification of the separate multi-bead complexes.
Identifying the
separate multi-bead complexes includes enumerating the separate multi-bead
complexes.
In some implementations, attaching two or more beads to each unit of the
chemical component
includes binding two or more attachment units to each unit of the one or more
units of the
chemical component, each of the attachment units also being attached to one or
more beads, such
that each multi-bead complex includes two or more beads, two or more
attachment units, and the
unit of the chemical component. In some implementations, the attachment units
include
antibodies. In some implementations, the attachment units include capsid
proteins or other
antigens from a pathogen, and the units of the chemical component include
antibodies to the
pathogen. In some implementations, the pathogen includes a virus. In some
implementations, at
least two of the two or more attachment units are different from one another.
In some
implementations, the two or more attachment units bind at different locations
of a unit of the
chemical component.
Implementations may include one or a combination of two or more of the
following features. At
least two of the two or more beads of a multi-bead complex have the same
reflective, refractive,
and transmissive characteristics. At least two of the two or more beads of a
multi-bead complex
have different reflective, refractive, or transmissive characteristics or
combinations of them for
the light originating at the light source. The different reflective,
refractive, or transmissive
characteristics include at least one of colors of the beads, sizes of the
beads, shapes of the beads,
and birefringence of the beads. Each unit of the chemical component includes
an antibody of a
pathogenic virus. Placing the sample on the surface of the image sensor
includes forming a
monolayer of the sample on the surface. Placing the sample on the surface of
the image sensor
includes confining the sample between the surface of the image sensor and a
second surface
opposite the surface of the image sensor. The method includes identifying, in
at least one of the
images of the sample, separate individual beads based on light reflected by,
refracted by, or
transmitted by each of the separate individual beads, and based on a proximity
of each of the
separate individual beads to other beads.
In general, an aspect of the disclosure is an apparatus including an image
sensor having an array
of light sensitive elements at a surface of the image sensor, and one or more
computing
processors communicatively coupled to the image sensor, the one or more
computing processors
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configured to perform operations including: receiving, from the image sensor,
data representative
of one or more images of a sample situated at the surface of the image sensor,
the sample
including one or more multi-bead complexes, each multi-bead complex including
two or more
beads attached to a unit of a chemical component, in which the one or more
images are based on
.. light originating at a light source and received at the image sensor, the
received light including
light reflected by, refracted by, or transmitted through the beads of the
multi-bead complexes;
and identifying, in at least one of the images of the sample, separate multi-
bead complexes, the
identifying of the separate multi-bead complexes including associating the two
or more beads of
each of the multi-bead complexes based on close proximity to one another.
Implementations may include one or a combination of two or more of the
following. The
operations include identifying a presence of the chemical component based on
the identification
of the separate multi-bead complexes. The operations include identifying a
level of the chemical
component based on the identification of the separate multi-bead complexes.
Identifying the
separate multi-bead complexes includes enumerating the separate multi-bead
complexes.
.. In some implementations, each multi-bead complex includes two or more
attachment units
bound to a unit of the chemical component, each of the attachment units also
being attached to
one or more beads, such that each multi-bead complex includes two or more
beads, two or more
attachment units, and the unit of the chemical component. In some
implementations, the
attachment units include antibodies. In some implementations, the attachment
units include
.. capsid proteins or other antigens from a pathogen, and the units of the
chemical component
include antibodies to the pathogen. In some implementations, the pathogen
includes a virus. In
some implementations, at least two of the two or more attachment units are
different from one
another. In some implementations, the two or more attachment units bind at
different locations of
a unit of the chemical component.
Implementations may include one or a combination of two or more of the
following. At least two
of the two or more beads of a multi-bead complex have the same reflective,
refractive, and
transmissive characteristics, and the one or more processors are configured to
detect the
reflective, refractive, and transmissive characteristics. At least two of the
two or more beads of a
multi-bead complex have different reflective, refractive, or transmissive
characteristics or
.. combinations of them for the light originating at the light source, and the
one or more processors
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are configured to detect the reflective refractive, and transmissive
characteristics. The different
reflective, refractive, or transmissive characteristics include at least one
of colors of the beads,
sizes of the beads, shapes of the beads, and birefringence of the beads. Each
unit of the chemical
component includes an antibody of a pathogenic virus. The apparatus includes a
second surface
opposite the surface of the image sensor, the second surface configured to
confine the sample
between the second surface and the surface of the image sensor. The second
surface is
configured to form a monolayer of the sample between the second surface and
the surface of the
image sensor. The apparatus includes a light source. The operations include
enumerating, in at
least one of the images of the sample, separate individual beads based on
light reflected by,
refracted by, or transmitted by each of the separate individual beads, and
based on a proximity of
each of the separate individual beads to other beads.
These and other aspects, features, implementations, and advantages (1) can be
expressed as
methods, apparatus, systems, components, program products, business methods,
means or steps
for performing functions, and in other ways, and (2) will become apparent from
the following
description and from the claims.
Description
Figures 1, 2, 3, 5, and 6 are schematic views of chemical analysis of samples.
Figure 4 is a graph of a standard curve.
Here we describe a sample analysis technology that in some implementations can
perform a
chemical analysis of a sample of whole blood alone or in combination with a
CBC directly at a
point of care within a few minutes at low cost using a small portable easy-to-
use, relatively
inexpensive sample analysis device. In some uses, because of its small size
and low cost, the
sample analysis device can be reproduced in large numbers and distributed to
many locations
within one or more healthcare, residential, industrial, or commercial
locations. In some
applications, many units of the sample analysis device can be distributed and
used in the field
including at locations where equipment for sample analysis (for example, blood
chemistry or
CBC) is otherwise unavailable or prohibitively expensive.
We use the term "point-of-care" broadly to include, for example, any location
in close physical
proximity to a patient or other person to whom healthcare is being provided.
In many cases,
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point-of-care refers to services provided in the physical presence of a
patient, for example, in the
same room or building or at the same place or within a short distance.
Although much of the discussion below refers to applications of the sample
analysis technology
to chemical analysis of whole blood drawn from a human or other animal, the
sample analysis
technology can also be applied to a wide range of contexts in which a sample
(which may, but
need not, be a biological sample) contains chemical components of interest
(such as molecules or
ions) and that may not involve counting and may or may not include particles,
units, or other
elements of one or more kinds that are to be counted.
We use the term "sample" broadly to include, for example, any fluid or other
mass or body of
material that contains one or more analyzable chemical components and may or
may not also
contain one or more countable units of one or more types. The countable units
may in some cases
be opaque, translucent, or otherwise non-transparent to incident light. The
analyzable chemical
components may in some instances be transparent, translucent, or otherwise non-
opaque to
incident light. In some examples, the sample is whole blood containing
countable blood cells of
different types and also containing analyzable chemical components such as
molecules or ions,
to name two.
We use the term "chemical components" broadly to include, for example,
chemical compounds,
ions, molecules, and other constituents of a sample that may not be present in
a form of
discernible (e.g., visible) countable units.
We use the term "unit of a chemical component" broadly to include, for
example, a single unit of
a chemical component such as a single molecule, ion, or other constituent. In
typical samples,
there are many units of a given type of chemical component, for example, many
molecules of a
chemical compound.
We use the term "countable units" broadly to include, for example, elements
present in a sample
that are discrete, discernible, visible, identifiable, and subject to
enumeration. Typically,
countable units are not transparent. In the case of whole blood, the countable
units can include
blood cells of different types.
We use the term "chemical analysis" broadly to include, for example,
identification and
quantification (e.g., determination of the level) of chemical components of
one or more types in
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the sample. In some cases, chemical analysis can include identifying the
presence of one or more
molecules of one or more types and characterizing the amount, volume, or
percentage of each of
the types of molecules in the sample or in a particular volume of the sample.
As noted earlier, although the sample analysis technology has a broader range
of applications, for
convenience we sometimes discuss particular examples in which the sample
includes whole
blood or components of whole blood.
We use the term "whole blood" broadly to include, for example, blood in its
original form drawn
from a human or other animal. Whole blood includes countable units such as
blood cells and
blood plasma that includes chemical components. As described in the Wikipedia
entry titled
"Blood plasma" blood plasma is "a yellowish liquid component of blood that
normally holds the
blood cells in whole blood in suspension. In other words, it is the liquid
part of the blood that
carries cells and proteins ... It is mostly water (up to 95% by volume), and
contains dissolved
proteins (6-8%) (e.g. serum albumins, globulins, and fibrinogen), glucose,
clotting factors,
electrolytes (Nat, Ca', Mg', HCO3-, Cl-, etc.), hormones, carbon dioxide
(plasma being the
main medium for excretory product transportation) and oxygen." Clotting
factors include
molecules such as plasminogen and prothrombin that participate in clot
formation.
We use the term "blood cells" broadly to include, for example, red blood cells
(erythrocytes),
white blood cells (leukocytes), rare blood cell types, ambiguous blood cell
types, and platelets
(thrombocytes).
As shown in figure 1, typical automated techniques 10 for chemical analysis of
blood use
fluorescence-based sandwich immunoassay techniques to identify and quantify
acellular
chemical components 12, for example, molecules of one or more chemical
components in blood
plasma 14. The "filling" of the "sandwich" in fluorescence-based sandwich
immunoassay is, for
example, molecules 16 of a given target chemical component in the blood
plasma. Each of the
molecules is, in effect, sandwiched 18 as a result of adding two types 20, 22
of antibodies to the
blood sample. The antibodies of one type 20 are known to bind specifically to
one location 24 on
the target molecules and serve as "capture antibodies" in the sense that they
provide a known
"base" at which the target molecules are held. The antibodies of the other
type 22 serve as
"detection antibodies" and are also known to bind specifically to the target
molecules, but to a
.. different location 26 on the target molecules. In some examples, the
capture antibodies are fixed,
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say, to a surface 28 and literally "capture" the target molecules and hold
them at a particular
location on the surface. The detection antibodies are typically marked by
fluorescent molecules
30 attached to them.
Once the target molecules have been captured, that is, bound to the capture
antibodies, high
intensity excitation light 32 illuminates the sample in one wavelength band
causing much lower
intensity light to be emitted 34 from the attached fluorescent molecules in a
different, typically
longer, fluorescence wavelength band. The emitted light is sensed by a light
detector 36 (after
being passed through a filter 38 to block the much higher intensity excitation
light). The light
detector is highly sensitive to the presence and intensity level of the
relatively low intensity
fluorescence wavelength band light and can therefore generate signals
indicating the
fluorescence intensity and in turn the amount of the target chemical component
present in the
sample.
The fluorescence sandwich technique can be used to identify and quantify
different target
chemical components of blood simultaneously by using different appropriate
pairs of capture
antibodies and suitably labelled (by fluorescent molecules) detection
antibodies. In some
implementations of such multiplexing, the different capture antibodies are
attached at different
locations to a fixed surface as a way to differentiate the different target
molecules based on their
locations at the fixed surface. In some implementations, the target molecules
remain dissolved or
suspended in the sample and the different capture antibodies are marked using
fluorescent beads
(for example, Luminex beads) that produce fluorescence light in different
wavelength bands, or
different combinations of the bands, as a way to differentiate the different
types of target
molecules without regard to their locations in the sample.
As discussed later, in some implementations of the sample analysis technology,
chemical
analysis is combined with a contact monolayer non-fluorescence imaging
technique for
performing a complete blood count (CBC). For several reasons, the standard
fluorescence
sandwich technique just described is not optimally compatible with the contact
monolayer non-
fluorescence CBC technique. One reason is that, in the contact CBC technique,
the blood sample
is typically in direct contact with a light-sensitive surface of an image
sensor which precludes the
inclusion of a filter element between the surface and the sample to block the
high intensity
excitation light. A second reason is that the contact CBC technique is not
readily compatible with
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washing and other processing steps (one of which involves removing non-
transparent blood cells
from the sample) generally required in fluorescence sandwich immunoassay
techniques. The
washing and processing steps cannot be easily applied if the same whole blood
sample used for
the sample analysis technique is to be used also for the contact CBC
technique. [Yet, as will be
discussed later, because the contact CBC technique is based on the use of a
monolayer of blood,
portions of the monolayer are free of blood cells and contain only light-
passing blood plasma.
Therefore, although the entire area of the image sensor may not be suitable
for chemical analysis
of the target molecules because of the presence of blood cells, some portions
of the area of the
image sensor are suitable for the sample analysis technique even with whole
blood.] A third
reason why the fluorescent sandwich technique is not optimally compatible with
the contact
CBC technique described above is that the small size pixels of the high-
resolution image sensor
do not provide as adequate low-light sensitivity to detect the low intensity
emitted fluorescence
light as can a larger-area light detector.
The sample analysis technology that is described here can be used
independently to perform
chemical analysis of whole blood or can be used to perform chemical analysis
of whole blood in
combination with or to supplement (simultaneously or sequentially) a contact
CBC technique
that uses the same sample and light from the same light source. As a result,
both the contact CBC
technique and the blood chemical analysis can be performed quickly at
essentially the same time
on a tiny sample of whole blood (for example, a sample of less than 50
microliters or less then 15
microliters or less then 5 microliters) at the point-of-care using a small
inexpensive device.
Although we often discuss examples in which the chemical analysis is performed
on whole
blood, the sample analysis technology can be applied to raw whole blood or to
whole blood that
has been processed to alter or adjust or remove or supplement chemical
components or to whole
blood from which some or all of the blood cells have been removed, including
blood plasma.
We use the term "contact CBC technique" broadly to include, for example, any
technique in
which blood cells of one or more types are identified and counted in a sample
that is in contact
with (e.g., within a near-field distance of) a surface of an image sensor.
Additional information
about contact CBC techniques can be found in one or more of United States
patent publications
2016/0041200, 2014/0152801, 2018/0284416, 2017/0293133, 2016/0187235, and
United States
patents 9,041,790, 9,720,217, 10,114,203, 9,075,225, 9,518,920, 9,989,750,
9,910,254,
9,952,417, 10,107,997, all of which are incorporated here by reference.
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Referring to figure 2, in some implementations of the sample analysis
technology, a monolayer
100 of whole blood is situated between a surface 102 of a high resolution
image sensor 104 at
which an array of photosensitive elements (e.g., pixels) 106 are exposed and a
corresponding
surface 108 of a lid 110, to form a monolayer having a known volume defined by
its length,
width, and thickness 112 between the surface 102 and the surface 108. Examples
of structures
and techniques for forming such a monolayer are described in one or more of
United States
patent publications 2016/0041200, 2014/0152801, 2018/0284416, 2017/0293133,
2016/0187235,
and in United States patents 9,041,790, 9,720,217, 10,114,203, 9,075,225,
9,518,920, 9,989,750,
9,910,254, 9,952,417, 10,107,997õ all of which are incorporated here by
reference.
We use the term "high-resolution" broadly to include, for example, an image
sensor that has a
pixel spacing in one or both of two dimensions that is smaller than 5 [tm, or
3 [tm, or 1 [tm, or
sub-micron, for example.
We use the term "monolayer" broadly to include, for example, a volume of a
sample that has a
thickness no greater than the thickness of a particular type of unit in the
sample, such as blood
cells, so that across the monolayer two units cannot be stacked in the
dimension defined by the
thickness. In the case of a whole blood sample, the thickness of the monolayer
could be in the
range of 1 micrometer to 100 micrometers.
Light 120 from a light source 122 illuminates the monolayer 100. Portions 124
of the light may
pass through the sample monolayer and be received by photosensitive elements
126 in the array
128 of the image sensor. Portions 130 of the light may be reflected or
refracted by components
131 of the monolayer and the reflected or refracted light may be received by
photosensitive
elements in the array. Portions 132 of the light may be transmitted through
components of the
monolayer and the transmitted light may be received by photosensitive elements
in the array;
portions of the light may be absorbed by components of the monolayer. As
discussed later, the
components of the monolayer can include countable units, chemical components,
beads, and
other elements.
The light source can be configured or controlled or both to provide
illuminating light in one or
more selected wavelength bands and combinations of them. A wide variety of
types of light
sources and combinations of them can be used, for example, LEDs, LED panels,
organic LEDs,
fluorescent panels, incandescent lamps, ambient illumination, arrays of
monochrome LEDs,
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arrays of narrowband sources such as red, green, and blue LEDs or lasers, a
miniaturized color
display such as a liquid crystal or organic LED (OLED) display or an RGB laser
color projector.
Using the light that originates at the light source and passes through, is
reflected or refracted by,
or is transmitted through the monolayer, the image sensor captures one or more
images of the
monolayer including countable units of various types (for example, blood
cells) and chemical
components that are detectable (either in their native condition or as a
result of being marked as
discussed later). One or more of the captured images are processed by one or
more processors or
other image processing components 113 to produce information 133 about the
whole blood
sample including, for example, a CBC or a chemical analysis or both of the
countable units and
chemical components. Among other things, the resulting information can include
a count of red
blood cells and their hemoglobin content.
The CBC information can be generated by identifying and counting in the
captured images the
number of countable units of each type in the sample. Additional information
about CBC
techniques and about imaging using contact image sensors can be found, for
example, in United
States patent publications 2016/0041200, 2014/0152801, 2018/0284416,
2017/0293133,
2016/0187235, and in United States patents 9,041,790, 9,720,217, 10,114,203,
9,075,225,
9,518,920, 9,989,750, 9,910,254, 9,952,417, 10,107,997, all of which are
incorporated here by
reference.
As shown in figure 3, a monolayer 140 of whole blood (such as the same
monolayer of whole
blood used for the contact CBC technique) can be used for chemical analysis of
various chemical
components 142, 144 of the whole blood. For this purpose, individual units of
the different types
of chemical components of the whole blood monolayer sample can be treated as
fillings of
sandwiches 148 similar to the fluorescent sandwiches. However, in
implementations of the
sample analysis technology described here, capture antibodies 150, 152 and
detection antibodies
154, 156 are attached to beads 158, 160, 162, 164 that need not have
fluorescent properties and
are directly visible or otherwise detectable using light that originates at
the light source and
passes through, is reflected or refracted by, or is transmitted through the
monolayer or
components of the monolayer. The resulting light is received by light-
sensitive elements (e.g.,
pixel) 166 arrayed in the image sensor 168. (Unlike fluorescence techniques,
the light source is
not within the monolayer sample but is external to it.)
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Using the received light (in some cases, the same received light used for the
contact CBC
technique), the image sensor captures one or more images of the monolayer
sample. One or more
processors 170 or other image processing devices process the one or more
received images and
apply a variety of techniques to identify the presence of and determine the
level (e.g., quantity,
amount, volume, percentage) of each of the chemical components in the sample.
The beads 158, 160, 160, 162 to which the antibodies 150, 152 and 154, 156 are
attached need
not have fluorescent properties. The beads can have characteristics that are
detectable, visible, or
otherwise discernible based on light from the light source that is reflected
from, refracted by, or
passes through them. We sometimes refer to such beads as "direct indicator
beads". The direct
indicator beads can take the form of what are sometimes call microbeads in
reference to their
small size. Microbeads have sizes typically in the range of 0.5 to 500
micrometers.
We use the term "direct indicator beads" (or sometimes simply "beads") broadly
to include, for
example, any tag, marker, or other indicator device or indicator
characteristic that can be
attached to or associated with a chemical component of a sample and is
identifiable at a sensor
using received light that was incident on and reflected or refracted by or
transmitted through the
indicator device or characteristic. In some cases, direct indicator beads can
take the form of small
grains, particles, beads, spherules, or other elements, and combinations of
them, and can be of a
variety of shapes, sizes, materials, and colors.
To determine the presence of units of chemical components in the sample, the
processor analyzes
the images to detect directly discernible characteristics of beads and
complexes of two or more
beads that are revealed by light originating from the light source and
reflected from, refracted by,
or transmitted through the beads to the surface of the image sensor.
We use the term "directly discernible characteristics" of beads and complexes
of beads broadly
to include, for example, any quality, attribute, or other trait that can be
detected, determined, or
derived from light that originated at a light source and was reflected from,
refracted by, or
transmitted through the beads. Directly discernible characteristics could
include color, size,
texture, birefringence, or shape, or combinations of them, for example.
We use the term "complexes of beads" broadly to include, for example, two or
more beads that
can be associated with one another because they are attached to a unit in a
sample, such as a
molecule or other chemical component. Typically, the two or more beads of a
complex are
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detectable in constant close proximity (e.g., touching) one to another. In
some cases, the two or
more beads of a complex are detectable because they have two or more
predetermined different
directly discernible characteristics. For example, two beads of a complex may
have two specific
different colors that are discernible by processing the images from the image
sensor.
We use the word "attach" to include both direct and indirect attachment. For
example, two
particles, units, or other elements may be attached directly (e.g., in contact
and bound) to one
another, or indirectly (e.g., attached to one another by an attachment unit
bound to each of the
two particles, units, or other elements).
We use the term "attachment unit" broadly to include, for example, any
antibodies (e.g., an
antibody directed against a cluster-of-differentiation cell surface antigen if
the target unit is a
specific cell type), capsid proteins or other antigens from a pathogenic virus
(e.g., if the target
unit is an antibody to the pathogenic virus, indicating prior exposure to the
pathogenic virus),
and other binding molecules and structures suitable for binding or attachment
(e.g., direct
attachment) to a unit of a chemical component.
.. The sample analysis technology that we describe here can be applied in a
variety of different
modes.
In some examples of one such mode, which we sometimes call the complexed-beads
mode, the
chemical components remain dissolved or suspended in the sample. A capture
antibody and a
detection antibody, each coupled to a separate direct indicator bead, bind
simultaneously to the
two different locations on a given target molecule or other unit of a target
chemical component to
form a complex of two beads (i.e., a doublet). [Because each direct indicator
bead has more than
one of its particular (capture or detection) antibody bound to its surface, a
bead may participate
in more than one such complex simultaneously, forming a triplet or higher-
order bead complex.]
By processing one or more images captured by the image sensor, it is possible
to identify those
beads present in doublets or higher-order complexes, and thus associated with
the chemical
component. By determining the proportion of complexed beads to the total
number of beads
(complexed and singleton, that is, uncomplexed) identified in the sample, it
is possible to
determine the level or amount or quantity or concentration of the target units
(e.g., molecules) of
the chemical component in the sample.
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It is true that identified singleton beads are not necessarily beads unbound
to the target molecule,
because in some cases only the capture antibody or the detection antibody, but
not both, may
have bound to the target molecule.
However, under constant incubation conditions and provided that the
concentrations of bead-
coupled capture antibodies and bead-coupled detection antibodies in the sample
are constant and
their ratio is known, it is possible empirically to establish a "standard
curve" that represents the
relationship between the bead complex index (that is, the proportion of
complexed beads to total
beads identified by the device) and the concentration of the target molecule.
This has been done experimentally for prolactin to generate the standard curve
shown in figure 4.
Using the standard curve, it is possible to determine the otherwise unknown
concentration of
prolactin in a sample by determining the bead complex index under identical
incubation
conditions.
In some implementations, the same beads can be used to mark both the capture
antibodies and
the detection antibodies that will bind to given units of the chemical
component. In some
implementations, by using complexes of beads having different directly
discernible
characteristics for capture antibodies and detection antibodies that are to be
attached to the units
of different chemical components it is possible to multiplex the process of
detecting the presence
and levels of the different chemical components at the same time. Multiplexing
can be achieved
by using beads having different colors, sizes, shapes, textures, or other
directly discernible
characteristics.
As shown in figure 5, in some implementations, the capture antibodies 200 are
irreversibly
bound to a fixed surface 202, for example different types of capture
antibodies are bound as
spots 206 in known corresponding locations in an array 204 on the fixed
surface. In such
implementations the capture antibodies need not have direct indicator beads
attached to them, but
the detection antibodies would have direct indicator beads attached to them.
The fixed surface
could be the surface 108 of the lid 110 that faces the surface 102 of the
image sensor 104 and
defines a gap occupied by the monolayer 100 of the sample. When the monolayer
of the sample
is in the gap and in contact with the printed spots in the array of capture
antibodies, respective
chemical components in the sample will bind to respective capture antibodies,
based on the type
of the chemical components, in positions defined by the locations of the
printed spots in the
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array, and can at the same time bind to detection antibodies coupled to direct
indicator beads.
Images captured using incident light that passes through the monolayer and is
reflected, refracted
or transmitted by the direct indicator beads can then be processed to identify
and determine the
amounts of different types of chemical components based on the imaged
locations of the beads
attached to the detection antibodies. This technique of chemical analysis can
be used separately
or in combination with the contact CBC technique discussed earlier.
In some implementations, a combination of the location-based chemical analysis
technique and
the in-solution or in-suspension (that is, non-location-based, complexed-beads
mode) chemical
analysis technique could be used.
In order to use these chemical analysis techniques in combination with the CBC
technique in a
point-of-care setting, steps must be taken to impart the bead-coupled
antibodies to the sample
before it is loaded onto the image sensor surface. One approach would be to
pass the sample of
blood taken from the patient through a tube where dried bead-coupled
antibodies are solubilized
by the blood and allowed to bind with the target molecules. Then the prepared
sample can be
placed on the sensor surface. Another approach would be to deposit the bead-
coupled antibodies
onto the surface 108 of the lid 110 (in some cases in addition to bead-free
capture antibodies
irreversibly bound at specific locations of the lid) so that they are
solubilized when the lid
encounters the blood sample in forming the monolayer.
In some implementations, the target unit of the chemical component includes at
least one of an
antigen, a hormone, a biomarker, a drug, a viral capsid, a pathogen-directed
antibody (for
example, a virus-directed antibody), an oligonucleotide, or another molecule,
cell, or particle.
In some implementations, a bead is bound to an attachment unit, and the
attachment unit is
bound to the target unit of the chemical component. In the example of Figure
3, attachment units
150 and 154 are bound to a first target unit 142 of a first chemical
component, and attachment
units 152 and 156 are bound to a second target unit 144 of a second chemical
component. Beads
158 and 160 are bound to attachment units 150 and 154, respectively, to form a
multi-bead
complex 143 that includes beads 158 and 160, attachment units 150 and 154, and
target unit 142.
Beads 162 and 164 are bound to attachment units 152 and 156, respectively, to
form a multi-bead
complex 145 that includes beads 162 and 164, attachment units 152 and 156, and
target unit 144.
In some implementations, it is the proximity of the beads 162 and 164 to one
another or the
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constancy of the proximity or both that allows for the identification of the
multi-bead complex.
The proximity can be measured in terms of absolute distance, or proportion of
the dimension of
one or more of the beads, for example.
The attachment units 150, 152, 154, 156 can be, but need not be, detection
antibodies or capture
antibodies. In addition to, or alternatively to, antibodies, the attachment
units 150, 152, 154, 156
may include a capsid protein or other antigen of a pathogenic virus. The
attachment units 150,
154 may be different from one another.
The target units 142 and 144 of the chemical component may include at least
one of an antigen, a
hormone, a biomarker, a drug, a viral capsid, a pathogen-directed antibody
(for example, a virus-
directed antibody), an oligonucleotide, or another molecule, cell, or
particle. The attachment
units 150, 152 (for example) may include proteins of a virus, the proteins
binding to an antibody
142 of the virus.
In some implementations, the beads 158, 160 are different from one another in
one or more
characteristics such as size, color, shape, surface properties, translucency,
weight, and
combinations of them.
As shown in Figure 6, in some implementations, an attachment unit 306 (for
example, a capture
unit, capture particle, or other capture element) is bound at a known location
308 on a surface
310 (e.g., a surface of an image sensor, or a surface of a lid). The surface
310 may include an
array of known locations 312, each known location corresponding to a known
type of attachment
unit (not shown, except for 306). The attachment unit 306 is bound to a target
unit 304 of a
chemical component, which is bound to an attachment unit 302. The attachment
unit 302 is
bound to a direct indicator bead 300. As described in reference to Figure 5,
because the
attachment unit 306 is of a known type (e.g., a type known to bind to the
target unit 304 of the
chemical component) and in a known location, imaging of the direct indicator
bead 300 and
determination of the known location 308 may be used to determine an amount or
presence or
both of the chemical component.
The attachment units 302 and 306 can be, but need not be, antibodies, and can
include types of
attachment units as described above in reference to Figure 3. The target unit
304 of the chemical
component may include at least one of an antigen, a hormone, a biomarker, a
drug, a viral capsid,
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a pathogen-directed antibody (for example, a virus-directed antibody), an
oligonucleotide, or
another molecule, cell, or particle.
In some implementations, a capture bead includes an attachment unit. In some
implementations,
a surface (for example, the surface 310) includes an attachment unit.
Various choices of the target units and attachment units may be used in a
variety of applications,
such as cytometry, in vitro diagnostics, environmental analysis, multiplex
biochemical assays,
serology, and gene expression and combinations of them.
In an example of serology applied to a sample of blood from a patient, beads
are bound to
recombinant viral proteins of an infectious virus. The recombinant viral
proteins bind to
antibodies of the infectious virus within the sample. Complexes of two or more
of the beads
associated with an antibody of the infectious virus are identified or
enumerated or both, and
results of the identification or enumeration or both (potentially in concert
with the identification
or enumeration or both of single un-complexed beads) are used to determine a
presence or level
or both of the antibody of the infectious virus, as described earlier. Based
on the determined
presence or level or both of the antibody of the infectious virus, past
exposure by the patient to
the infectious virus can be identified. Samples besides blood may be used in
addition to or
instead of blood.
Other implementations are also within the scope of the following claims.
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