U.S. patent application number 14/359343 was filed with the patent office on 2014-09-11 for plasmonic substrates for metal-enhanced fluorescence based sensing, imaging and assays.
This patent application is currently assigned to UNIVERSITY OF MARYLAND, BALTIMORE. The applicant listed for this patent is Joseph Lakowicz, Henryk Szmacinski. Invention is credited to Joseph Lakowicz, Henryk Szmacinski.
Application Number | 20140256593 14/359343 |
Document ID | / |
Family ID | 48470345 |
Filed Date | 2014-09-11 |
United States Patent
Application |
20140256593 |
Kind Code |
A1 |
Szmacinski; Henryk ; et
al. |
September 11, 2014 |
PLASMONIC SUBSTRATES FOR METAL-ENHANCED FLUORESCENCE BASED SENSING,
IMAGING AND ASSAYS
Abstract
Techniques for metal enhanced fluorescence include determining a
calibration curve that relates concentration of a particular
analyte to at least one of intensity or lifetime of fluorescent
emissions at a functionalized substrate in response to incident
light, for a plurality of known concentrations of the particular
analyte mixed with a reagent. The functionalized substrate
comprises a plasmonic substrate and a bioactive target molecule
that has an affinity for the particular analyte. The reagent
comprises a detection molecule. A concentration of the particular
analyte in a sample is determined directly from the calibration
curve and measurements, in response to the incident light, of at
least one of intensity or lifetime of fluorescent emissions at the
functionalized substrate in contact with the sample and
reagent.
Inventors: |
Szmacinski; Henryk;
(Mariottsville, MD) ; Lakowicz; Joseph; (Ellicott
City, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Szmacinski; Henryk
Lakowicz; Joseph |
Mariottsville
Ellicott City |
MD
MD |
US
US |
|
|
Assignee: |
UNIVERSITY OF MARYLAND,
BALTIMORE
Baltimore
MD
|
Family ID: |
48470345 |
Appl. No.: |
14/359343 |
Filed: |
November 23, 2012 |
PCT Filed: |
November 23, 2012 |
PCT NO: |
PCT/US12/66451 |
371 Date: |
May 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61562667 |
Nov 22, 2011 |
|
|
|
61592851 |
Jan 31, 2012 |
|
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Current U.S.
Class: |
506/9 ; 359/884;
435/7.1; 436/501; 506/18; 977/773 |
Current CPC
Class: |
B82Y 15/00 20130101;
B82Y 20/00 20130101; G01N 33/54373 20130101; G01N 21/274 20130101;
G01N 33/553 20130101; G01N 33/56972 20130101; G02B 5/008 20130101;
G01N 33/6863 20130101; C12Q 1/02 20130101; G01N 33/54346 20130101;
G01N 21/648 20130101; G01N 21/554 20130101; G01N 21/6408 20130101;
Y10S 977/773 20130101 |
Class at
Publication: |
506/9 ; 359/884;
436/501; 435/7.1; 506/18; 977/773 |
International
Class: |
G01N 33/543 20060101
G01N033/543; G01N 33/68 20060101 G01N033/68; G01N 33/569 20060101
G01N033/569; G02B 5/00 20060101 G02B005/00 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with government support under Grant
No. CA147975 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1. A method comprising: providing a functionalized substrate
comprising a plasmonic substrate that is functionalized with a
bioactive target molecule that has an affinity for a particular
analyte; providing a reagent comprising a detection molecule for
the particular analyte; determining a calibration curve that
relates concentration of the particular analyte to at least one of
intensity or lifetime of fluorescent emissions at the
functionalized substrate in response to incident light for a
plurality of known concentrations of the particular analyte mixed
with the reagent, contacting a sample and the reagent to the
functionalized substrate; obtaining measurements of at least one of
intensity or lifetime of fluorescent emissions at the
functionalized substrate in contact with the sample and reagent in
response to the incident light; and determining a concentration of
the particular analyte in the sample directly from the calibration
curve and the measurements.
2. A method as recited in claim 1, wherein the sample and reagent
are not rinsed from the functionalized substrate before obtaining
the measurements.
3. A method as recited in claim 1, wherein the sample comprises a
living cell and the analyte is secreted by the living cell.
4. A method as recited in claim 1, wherein the analyte is a
cytokine.
5. A method as recited in claim 1, further comprising; obtaining
second measurements of at least one of intensity or lifetime of
fluorescent emissions at the functionalized substrate in contact
with the sample and reagent in response to the incident light at a
later time; and determining a concentration of the particular
analyte in the sample at the later time directly from the
calibration curve and the second measurements.
6. A method as recited in one of claim 1 wherein: the particular
analyte comprises a plurality of different analytes; the
fluorescent emissions comprise emissions in a corresponding
plurality of emission wavelength bands associated with a
corresponding plurality of detection molecules comprising a
corresponding plurality of different fluorophores; and the
bioactive target molecule comprise a corresponding plurality of
different target molecules that have affinities for the plurality
of different analytes.
7. A method as recited in claim 6, wherein the plurality of
different analytes consists of four different analytes.
8. A method as recited in claim 3, further comprising determining
viability of the living cell.
9. A method as recited in claim 3, further comprising determining
phenotype of the living cell.
10. A method as recited in claim 1, wherein the plasmonic substrate
comprises: a layer of metal configured as a mirror to reflect
light, a layer of dielectric material disposed on the mirror; and a
layer of metal nanoparticles disposed on the layer of dielectric
material.
11. A method as recited in claim 10, wherein the layer of metal
nanoparticles has an optical density below about 1.
12. A method as recited in claim 10, wherein a thickness of the
dielectric layer is greater than about 20 nanometers.
13. A method as recited in claim 10, wherein a thickness of the
dielectric layer is in a range from about 20 nanometers to about 80
nanometers.
14. A method as recited in claim 10, wherein a thickness of the
dielectric layer is in a range from about 25 nanometers to about 80
nanometers.
15. A method as recited in claim 10, wherein a thickness of the
dielectric layer is selected to maximize fluorescent enhancement
for a particular fluorophore in the detection molecule.
16. A plasmonic substrate comprising: a layer of metal configured
as a mirror to reflect light, a layer of dielectric material having
a thickness greater than about 20 nanometers disposed on the
mirror; and a layer of metal nanoparticles disposed on the layer of
dielectric material.
17. A plasmonic substrate as recited in claim 16, wherein the layer
of metal nanoparticles has an optical density below about 1.
18. A plasmonic substrate as recited in claim 16, wherein a
thickness of the dielectric layer is in a range from about 20
nanometers to about 80 nanometers.
19. A plasmonic substrate as recited in claim 16, wherein a
thickness of the dielectric layer is in a range from about 25
nanometers to about 80 nanometers.
20. A fluorescence affinity assay kit for determining the quantity
of a particular analyte, comprising: a plasmonic substrate that
comprises a layer of metal nanoparticles affixed to a substrate; a
solution comprising a bioactive target molecule that has affinity
for a particular analyte, wherein the target molecule includes a
ligand for affixing to the plasmonic substrate; and a reagent
comprising at least one plurality of substantively identical
detection molecules, wherein the detection molecule comprises a
fluorophore, and the detection molecule has affinity for the
particular analyte.
21. A fluorescence affinity assay kit as recited in claim 20,
wherein: the plasmonic substrate further comprises a layer of metal
configured as a mirror to reflect light, and a layer of dielectric
material having a thickness greater than about 20 nanometers
disposed on the mirror; and the layer of metal nanoparticles is
disposed on the layer of dielectric material.
22. A computer-readable not-transitory medium carrying one or more
sequences of instructions, wherein execution of the one or more
sequences of instructions by one or more processors causes an
apparatus to perform the steps of: determining a calibration curve
that relates concentration of a particular analyte to at least one
of intensity or lifetime of fluorescent emissions at a
functionalized substrate in response to incident light for a
plurality of known concentrations of the particular analyte mixed
with a reagent, wherein the functionalized substrate comprises a
plasmonic substrate and a bioactive target molecule that has an
affinity for the particular analyte and the reagent comprises a
detection molecule; and determining a concentration of the
particular analyte in a sample directly from the calibration curve
and measurements of at least one of intensity or lifetime of
fluorescent emissions at the functionalized substrate in contact
with the sample and reagent in response to the incident light.
23. An apparatus comprising: a source of incident light, an optical
coupler configured to direct incident light onto a functionalized
substrate in contact with a mixture of a sample and a reagent,
wherein the functionalized substrate comprises a plasmonic
substrate and a bioactive target molecule that has an affinity for
a particular analyte and the reagent comprises a detection molecule
for the particular analyte; a detector configured to measure
fluorescent emissions from the functionalized substrate; at least
one processor; and at least one memory including one or more
sequences of instructions, the at least one memory and the one or
more sequences of instructions configured to, with the at least one
processor, cause the apparatus to perform at least the following,
determining a calibration curve that relates concentration of a
particular analyte to at least one of intensity or lifetime of
fluorescent emissions at the functionalized substrate in response
to the incident light for a plurality of known concentrations of
the particular analyte mixed with the reagent; and determining a
concentration of the particular analyte in a sample directly from
the calibration curve and measurements of at least one of intensity
or lifetime of fluorescent emissions at the functionalized
substrate in contact with the sample and reagent in response to the
incident light.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of Provisional Appln.
61/562,667, filed Nov. 22, 2011, and benefit of Provisional Appln.
61/592,851, filed Jan. 31, 2012, the entire contents of each of
which are hereby incorporated by reference as if fully set forth
herein, under 35 U.S.C. .sctn.119(e).
BACKGROUND OF THE INVENTION
[0003] In affinity assays, a known quantity of a labeled probe
competes with or binds to an unknown quantity of unlabeled analyte
at binding sites on a target molecule for which the analyte has an
affinity. The labeled probe that is bound to the target molecule
presents a different measurable phenomenon than the labeled probe
that is unbound. Calibration curves relate the presence or quantity
of the analyte to the relative amount of bound to unbound labeled
probe. The calibration curves are generated by measuring the
relative amounts of bound and unbound labeled probe in the presence
of known quantities of analyte. In sandwich binding assays, the
probe binds to the analyte that is bound to the target molecule. In
immunoassays, the analyte is an antigen and the target molecule is
an antibody.
[0004] Cytokines are important antigens that are secreted by cells
to promote a function of the cell. Cytokines control many
biological processes including inflammation and disease; therefore,
cytokine measurements are widely used in basic research and
diagnostics. Cytokines are small regulatory proteins and peptides
(with mass in a range from about 8 to about 30 kiloDaltons, kDa, 1
kDa=10.sup.3 daltons, 1 dalton=one twelfth of the mass of an
unbound neutral atom of carbon-12) that exhibit a wide range of
biological activities. Cytokines are released in unique profiles in
response to inflammation, infection, systemic infections such as
sepsis, chronic wound healing, and even as predictors of
mortality.
[0005] Affinity assays for cytokines secreted by a cell are
especially important but are beset by several challenges.
Physiological levels are often low; less than 10 picograms per
milliliter (pg/ml, where 1 pg=10.sup.-12 grams and 1 ml=10.sup.3
liters), which corresponds to a range from about 0.5 picoMolar (pM,
1 pM=10.sup.-12 Molar, 1 Molar=1 mole per liter) to about 5 pM. The
detection of cytokines is also complicated by spatial heterogeneity
of their secretion, rapid turnover and short life-time. Many
techniques involve the removal, degradation or death of cells that
secrete the cytokines, which make it difficult to assess the rate
of secretion or the phenotype associated with a cytokine
profile.
[0006] A majority of cytokine assays developed over the past decade
use specific anti-cytokine antibodies. Currently there are two
dominant technologies for the measurement of multiple cytokines in
biological samples including cell supernatants: multiplex sandwich
ELISA and bead-based assays.
[0007] One important aspect of biological and clinical studies is
finding correlation of cell phenotypes with the profile of
cytokines secreted by these cells. Technologies for single cell
assay include flow cytometry or intracellular cytokine cytometry
(ICC) and ELISPOT.
[0008] Flow cytometry is currently mostly used for measurements of
cell surface molecules and intracellular levels of cytokines. There
are several advantages of flow cytometry: single cell measurement;
multiple biomarker detection; and sorting of cell populations for
subsequent analysis. There are also several limitations of
cytometry: requirement for expensive equipment and trained
personnel; inability to measure secreted proteins from live cells;
and difficulties in performing analysis with samples of limited
cell number. For cytometry, the cell must be permeabilized and
treated with secretion inhibitors which, at the very least,
interfere with normal cell function.
[0009] ELISPOT assays permit the ex vivo identification of cells
actively secreting cytokines and can detect a single cell out of a
million, based on well-defined spots that clearly represent numbers
of cytokine-secreting cells. Several limitations of ELISPOT assays
include: no quantitative information on the level of secreted
cytokines; difficult multiplexing (even double cytokines is
difficult); and, a multi-step protocol that makes ELISPOT usage
difficult. To address the need for double cytokine profiling, a
fluorometric modification of ELISPOT (FLUOROSPOT) has been
reported. However, because of insufficient fluorescence signal, a
complex biochemical amplification is needed which is not convenient
for practical use. Simultaneous correlation of cell phenotypes (or
cell viability) with cytokine secretion is impossible to realize
with ELISPOT or standard fluorometric approaches because of the
required washing steps that remove the cells before the imaging
process.
SUMMARY OF THE INVENTION
[0010] The inventors have determined that improved techniques are
desirable for measuring rapidly cytokines and other analytes in
small quantities, or in the presence of living cells, or directly
without rinsing, or some combination. Techniques are provided for
plasmonic substrates for metal-enhanced fluorescence based sensing,
imaging and assays that alleviate one or more deficiencies of prior
art approaches.
[0011] In a first set of embodiments, a method includes direct
measurement of analytes in a sample, even at small concentrations.
The method includes providing a functionalized substrate comprising
a plasmonic substrate that is functionalized with a bioactive
target molecule that has an affinity for a particular analyte. The
method also includes providing a reagent comprising a detection
molecule for the particular analyte. The method further includes
determining a calibration curve that relates concentration of the
particular analyte to at least one of intensity or lifetime of
fluorescent emissions at the functionalized substrate in response
to incident light for a plurality of known concentrations of the
particular analyte mixed with the reagent. A sample and the reagent
are brought into contact to the functionalized substrate.
Measurements are obtained of at least one of intensity or lifetime
of fluorescent emissions at the functionalized substrate in contact
with the sample and reagent in response to the incident light. The
method includes determining a concentration of the particular
analyte in the sample directly from the calibration curve and the
measurements.
[0012] For example, in some embodiments of the first set, the
sample and reagent are not rinsed from the functionalized substrate
before obtaining the measurements. Furthermore, in some
embodiments, the sample comprises a living cell and the analyte is
secreted by the living cell. In still further embodiments, the
analyte is a cytokine.
[0013] In a second set of embodiments, an article of manufacture is
a plasmonic substrate that includes a layer of metal configured as
a mirror to reflect light. The substrate also includes a layer of
dielectric material having a thickness greater than about 20
nanometers disposed on the mirror. The substrate still further
includes a layer of metal nanoparticles disposed on the layer of
dielectric material.
[0014] In a third set of embodiments, an article of manufacture is
a fluorescence affinity assay kit that includes a plasmonic
substrate that comprises a layer of metal nanoparticles affixed to
a substrate. The kit also includes a solution made of a bioactive
target molecule that has affinity for a particular analyte, wherein
the target molecule includes a ligand for affixing to the plasmonic
substrate. The kit still further includes a reagent comprising at
least one plurality of substantively identical detection molecules.
The detection molecule includes a fluorophore; and, the detection
molecule has affinity for the particular analyte.
[0015] In other sets of embodiments, an apparatus or a
computer-readable not-transitory medium is configured to perform
one or more steps of the above method.
[0016] Still other aspects, features, and advantages of the
invention are readily apparent from the following detailed
description, simply by illustrating a number of particular
embodiments and implementations, including the best mode
contemplated for carrying out the invention. The invention is also
capable of other and different embodiments, and its several details
can be modified in various obvious respects, all without departing
from the spirit and scope of the invention. Accordingly, the
drawings and description are to be regarded as illustrative in
nature, and not as restrictive.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The present invention is illustrated by way of example, and
not by way of limitation, in the figures of the accompanying
drawings and in which like reference numerals refer to similar
elements and in which:
[0018] FIG. 1 is a block diagram that illustrates an example
sandwich assay, according to an embodiment;
[0019] FIG. 2 is a block diagram that illustrates an example glass
slide with multiple wells each containing a functionalized
substrate that includes a plasmonic substrate, according to an
embodiment;
[0020] FIG. 3 is a block diagram that illustrates an example
fluorescence measurement apparatus suitable for sensing or imaging
fluorescence such as for an assay upon which an embodiment of the
invention may be implemented;
[0021] FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D are block diagrams
that illustrates an example reference functionalized substrate
without a plasmonic substrate and with two example plasmonic
substrates, according to multiple embodiments;
[0022] FIG. 5 is a block diagram that illustrates an example
measurement of quantity of analytes from a single cell, according
to an embodiment;
[0023] FIG. 6 is a flow chart that illustrates an example method to
directly assay the quantity of an analyte in a sample without
removing the sample, according to an embodiment;
[0024] FIG. 7A, FIG. 7B and FIG. 7C are graphs that illustrates
example effects of annealing on shape and optical absorption of
nanoparticles in a plasmonic substrate, according to an
embodiment;
[0025] FIG. 8A and FIG. 8B are block diagrams that illustrate
example configurations in the vicinity of a substrate for which
optical fields are simulated to determine the effects of a
dielectric layer on fluorescence enhancement, according to an
embodiment;
[0026] FIG. 9A and FIG. 9B are graphs that illustrate the ability
of the simulation to match data, according to an embodiment;
[0027] FIG. 10A, FIG. 10B and FIG. 10C are graphs that illustrate
example enhancement of excitation light intensity based on
thickness of dielectric layer, according to various
embodiments;
[0028] FIG. 11A and FIG. 11B are graphs that illustrate example
enhancement of emission light intensity based on thickness of
dielectric layer, according to various embodiments;
[0029] FIG. 12A is a graph that illustrates example measured
enhancements of fluorescence intensity for an example plasmonic
substrate at multiple optical frequencies, according to various
embodiments;
[0030] FIG. 12B is a graph that illustrates example measured
lifetimes of fluorescent emission for an example plasmonic
substrate at multiple optical frequencies, according to various
embodiments;
[0031] FIG. 13 is a block diagram that illustrates example
cytokines of interest, according to various embodiments;
[0032] FIG. 14 is a graph that illustrates example differences
among lifetimes of fluorescence emissions in various layers in or
above the plasmonic substrate for three dyes, according to an
embodiment;
[0033] FIG. 15A and FIG. 15B are graphs that illustrate example
fluorescence enhancement calibration curves for two cytokines,
according to various embodiments;
[0034] FIG. 16A and FIG. 16B are images that illustrate example
spot quantification of two cytokines with and without washing,
according to various embodiments;
[0035] FIG. 17A, FIG. 17B and FIG. 17C are images that illustrate
example spot quantification of fluorescence in the presence of
cells for three concentrations of cells, respectively, according to
various embodiments;
[0036] FIG. 18A is a graph that illustrates example calibration
curve, according to an embodiment;
[0037] FIG. 18B is a graph that illustrates example advantages over
a prior art approach, according to an embodiment;
[0038] FIG. 19A, FIG. 19B and FIG. 19C are images that illustrate
example real-time measurement of time-dependent secretion of a
cytokine from a subset of cells in a population, according to
various embodiments;
[0039] FIG. 19D is a graph that illustrates example use of
calibration curve, according to an embodiment;
[0040] FIG. 19E is a graph that illustrates example measured time
series of cytokine secretion, according to an embodiment;
[0041] FIG. 20A, FIG. 20B and FIG. 20C, illustrate example enhanced
analysis of fluorescence data from both intensity and lifetime
measurements, according to an embodiment;
[0042] FIG. 21 is a graph that illustrates example correlation of
phenotype with profile of secreted cytokines, according to an
embodiment;
[0043] FIG. 22 is a block diagram that illustrates a computer
system upon which an embodiment of the invention may be
implemented; and
[0044] FIG. 23 illustrates a chip set upon which an embodiment of
the invention may be implemented.
DETAILED DESCRIPTION
[0045] Techniques are described for metal-enhanced fluorescence
(MEF) based sensing, imaging and assays. In the following
description, for the purposes of explanation, numerous specific
details are set forth in order to provide a thorough understanding
of the present invention. It will be apparent, however, to one
skilled in the art that the present invention may be practiced
without these specific details. In other instances, well-known
structures and devices are shown in block diagram form in order to
avoid unnecessarily obscuring the present invention.
[0046] The inventors have discovered a surprisingly stable increase
in intensity and decrease in lifetime of fluorescent emissions in
the presence of metal nanoparticles on a dielectric layer deposited
on a metallic mirror. These changes are shown to be related to
interactions between the plasmons on the nanoparticles and
fluorophores in their vicinity in the presence of excitation and
emission waveforms, as described in more detail below. A plasmon is
an oscillation of free electron density in a metal particle which
can form waves on metal surfaces with the same electric fields and
frequencies but shorter wavelengths than electromagnetic waves.
Metal surface plasmons with frequencies in the visible spectrum can
interact with light. Subsequently, the inventors determined how
these properties can be used to design a new family of assays for
direct quantification of analytes, such as cytokines, at even very
small concentrations, such as associated with cytokine secretion
from a single cell, and in real-time.
[0047] Some embodiments of the invention are described below in the
context of fluorescent immunoassays for cytokines in the presence
of silver nanoparticles on a substrate with a silicon dioxide
dielectric layer and a silver mirror. However, the invention is not
limited to this context. In other embodiments any biological entity
may be the analyte and any molecule with analyte affinity may be
the target molecule and any metal may be used for the nanoparticles
with or without the dielectric layer or mirror or both and the
mirror may be made of any metal or other reflective surface.
Furthermore, any fluorophore may be used to label a detection
molecule used to determine binding of analyte to target
molecule.
1. Definitions
[0048] As used in this description, the following terms have the
meanings given here.
TABLE-US-00001 amino acids An organic molecule comprising both
carboxyl and amino groups that can form peptide bonds with
complementary groups on other amino acids. 22 amino acids comprise
all the proteins found in most living organisms. analyte a
component of a sample for which a quantity is to be determined,
including but not limited to a polymer, a ligand, an antigen, an
antibody, a protein, a peptide, DNA, RNA, oligonucleotide, a virus
or a bacterium. assay a method to determine the quantity (e.g., the
presence, absence, or concentration) of one or more components
called analytes in a test sample. assay kit a collection of
materials to be used in an assay. BSA-bt biotinylated bovine serum
albumin, an example target molecule. concentration a fraction of a
sample by weight or volume which is due to a component of the
sample. cytokine A short protein or peptide associated with one or
more cell functions or reactions to outside stimulation that serves
as one of the signaling molecules used in intercellular
communication. detection a molecule labeled with a fluorophore that
is used to detect binding of an molecule analyte to a target
molecule by binding to the analyte or by competing with the analyte
for binding sites on the target molecule. Also called a
probe-fluorophore conjugate or probe-dye conjugate. fluorophore a
functional group in a molecule which absorbs electromagnetic waves
at a specific wavelength and subsequently emits electromagnetic
waves at a different specific wavelength. Fluorophores include, but
are not limited to, fluoresceins, eosin, coumarines, rhodamines,
cyanines, benzophenoxazines, phycobiliproteins or fluorescent
proteins. functionalized a substrate that is conditioned to perform
a particular function by substrate deposition of layers of one or
more types of molecules, such as a glass slide coated with
bioactive molecules that facilitate fixing of an analyte to the
substrate. ligand a functional group in a molecule which binds to a
metal, generally involving formal donation of one or more of its
electrons. Metal-ligand bindings range from covalent bonds to
electrostatic attraction between ions (ionic bonding). light
electromagnetic (em) waves in a visible portion of the
electromagnetic spectrum, which includes wavelengths in air from
about 300 to about 800 nanometers (nm, 1 nm = 10.sup.-9 meters).
nanoparticles particles each having a dimension in a size range
from about 1 to about 1000 nanometers, nm. 1 nm = 10.sup.-9 meters.
plasmon an oscillation of free electron density in a metal particle
which can form waves on metal surfaces with the same electric
fields and frequencies but shorter wavelengths than incident
electromagnetic waves. Metal surface plasmons with frequencies in
the visible spectrum can interact with light. Plasmonic A substrate
that includes a layer of metal nanoparticles that form substrate
plasmons with frequencies in a spectral band of one or more
fluorophores probe a molecule that is used to detect binding of an
analyte to a target molecule by binding to the analyte or by
competing with the analyte for binding sites on the target molecule
(the portion of a detection molecule excluding the fluorophore).
Probes include, but are not limited to, a polymer, a ligand, an
antigen, an antibody, a protein, an oligomer, a protein, a peptide,
DNA, RNA or an oligonucleotide. probe- A detection molecule.
fluorophore conjugate protein A large molecule made up of a long
chain of amino acids. Shorter chains of amino acids are called
peptides or protein fragments. reagent substance or compound
consumed during a chemical reaction. solution a liquid mixture.
streptavidin-dye the protein streptavidin labeled with one or more
fluorophores, and thus conjugate an example detection molecule.
substrate a material on which a process is conducted target
molecule a molecule which has an affinity for a particular analyte.
Target molecules include, but are not limited to, a polymer, a
ligand, an antigen, an antibody, a protein, a peptide, DNA, RNA, an
oligonucleotide. Also called a capture molecule. test sample a
sample, such as a biological sample, with an unknown quantity of an
analyte
2. Overview
[0049] Sandwich assays using plasmonic substrates are presented
that provide unique capabilities suitable for the quantification or
temporal monitoring of cytokines and other analytes secreted by
living cells or otherwise present in small or temporally changing
concentrations. The amplified florescence by plasmonic structures
is called metal-enhanced fluorescence (MEF), and occurs when a
fluorophore is excited close to the surface of metal particles or
nanostructures, typically within a range from about 3 nm to about
50 nm. The amplification of fluorescence is due to an enhanced
excitation field (interaction of incident light with
nanoparticles), enhanced emissive properties of the fluorophore
(increased quantum efficiency of coupled fluorophore plasmon
system), and enhanced emission collection (light reflected or
directed into a detector). The mechanism of interaction of light
with metallic nanostructures is well understood and many
experimental and theoretical works have been published, including
their applications to biotechnology. Other previous approaches
explored individual metallic particles for biosensing
applications.
[0050] The inventors recognized that the geometrical configuration
of MEF is ideal for construction of surface-based bioassays where
the transduction signal originates from changes in the location of
a dye-labeled biomolecule. Binding of a dye-labeled antibody to the
surface-captured antigen will result in a dramatic increase in
intensity and decrease in fluorescence lifetime due to the MEF.
Thus, detection sensitivity of a typical sandwich-type immunoassay
can be improved proportionally to the amplified fluorescence. This
leads, in some embodiments, to direct readout without need for
rinsing off a reagent or sample.
[0051] In some embodiments, a MEFspot method involves the use of
plasmonic substrates that optically amplify a signal from
surface-bound fluorescent probes resulting in the ability to image
intensity and/or lifetime changes. The substantial improvements
over conventional approaches are substantially simplified
biochemical procedures and new capabilities. The simplified
procedure includes a one-step assay and the ability to use live
cells with detection sensitivity of ELISA and ELISPOT. New
capabilities include real-time monitoring of secreted proteins by
cells, their direct quantification, and multiplexing. In addition,
the MEFspot method provides the opportunity for detailed studies of
selected live cells (or cell clusters) using standard fluorescence
microscopy. The method has transformative potential in that that
new approaches can be undertaken for studies of cellular function
in a heterogeneous cell population, for example by selection of
relevant spots (cells) and their detailed studies in environments
that closely mimic their natural biological environments. Abilities
of MEFspot for detailed cellular studies can lead to better
understanding the pathways and mechanisms that underpin cytokine
release in broad range of cells.
[0052] In some embodiments, MEFspot is used with Fluorescence
Lifetime Imaging Microscopy (FLIM), which provides a broad range of
image processing with simple visualization of cell function
processes which can elucidate cellular mechanisms underlying cancer
initiation and progression as well as information about other
diseases. Such disciplines as cell physiology, immunology, and
cancer research are examples where the MEFspot could substantially
facilitate research and provide significant, transformative
scientific advancements. These advantages substantially broaden and
increase sensitivity compared to traditional cytokine
measurements.
[0053] FIG. 1 is a block diagram that illustrates an example
sandwich assay, according to an embodiment. FIG. 1 depicts a
portion of a functionalized substrate, including a glass substrate
110 and metal nanoparticles 122. The functionalized substrate also
includes fixed target molecules 132 for a particular analyte as the
fixed bioactive molecules. The functionalized substrate is in
contact with a covering solution 160.
[0054] In typical sandwich assays, the covering solution 160 is a
result of a three step process. First the functionalized substrate
is contacted to a test sample that includes analyte molecules 170
that are not labeled with a fluorophore. The contact is maintained
for sufficient time under conditions that allow the amount of
analyte binding to the fixed target molecules 132 to be
proportional to the amount of analyte in the test sample. Such
times and conditions are easily determined by routine
experimentation. Next, the functionalized substrate is washed to
remove excess unbound analyte from the test sample. Then the
functionalized substrate with bound analyte is contacted to a
solution of reagent. The reagent includes detection molecules 172.
Each detection molecule 172 includes a fluorophore 180 and a
molecule that binds to the analyte 170 at a site on the analyte
different from the site that binds the analyte to the fixed target
molecule 132. In some embodiments, the reagent is also rinsed and a
neutral rinsing buffer solution remains as the covering solution.
The combination of the functionalized substrata, sandwiched analyte
and covering solution in steady state is called a product of the
assay.
[0055] The product of the assay is exposed to excitation
electromagnetic waves at a specific wavelength that excites
fluorescence of the fluorophore 180. In the illustrated example,
the functionalized substrate and covering solution are exposed to
excitation incident light 190 indicated by dotted arrows. The
fluorophores in the detection molecules are excited by the incident
light and fluoresce, emitting light at a different specific
wavelength. The fluorophores on detection molecules that are free
in cover solution 160 emit electromagnetic waves with particular
properties. For example, a detection molecule in solution emits
solution emitted light 192 indicated by a single dot dash arrow.
The collection of these emissions has a first property value which
is associated with the particular detection molecule. In contrast,
the fluorophores on detection molecules that are bound to the
analyte that is in turn bound to the fixed target molecules 132
emit electromagnetic waves with a different value of some property
due to metal enhanced fluorescence (MEF). For example, detection
molecules bound to analytes bound to fixed target molecules 132
emit substrate emitted light 194 indicated by a double dot dash
arrow. For the sandwich assay to function successfully without
rinsing the reagent, the collection of these emissions has an
optical property value substantially different from the value of a
collection of the solution emitted light 192. For example, the
presence of nanoparticles 122 causes a change in lifetime or
intensity of the substrate emitted light 194 compared to the
solution emitted light 192, as described in more detail below.
[0056] One or more optical properties of the mixed solution and
substrate emitted light are measured using an apparatus like
apparatus 300 depicted in FIG. 3. The object 390 is the product of
the assay, i.e., the functionalized substrate contacting the
covering solution 160. A calibration curve is used in some
embodiments to determine the ratio of bound to free detection
molecules for a measured value of one or more optical properties.
Other calibration curves, as is well known in the art, are used to
determine a resulting analyte associated with such a ratio of bound
to free detection molecules. The resulting analyte is used to
determine the quantity (e.g., the presence, absence or
concentration) of analyte in the test sample.
[0057] FIG. 2 is a block diagram that illustrates an example glass
slide with multiple wells each containing a functionalized
substrate that includes a plasmonic substrate, according to an
embodiment. The substrate includes a glass substrate 210, with
several wells 212 formed by the application of an array of barriers
211 in the illustrated embodiment, but which may be etched in the
glass in other embodiments. The wells are used for separating
reactions on different portions of the substrate. Deposited in the
wells is a layer (often called a film) 220 of metal nanoparticles.
Deposited on the film of metal nanoparticles is a layer 230 of
bioactive molecules. In some embodiments, there are no wells and
the entire glass substrate is coated uniformly with a film 220 of
metal nanoparticles and layer 230 of fixed bioactive molecules.
[0058] Any material which does not unduly interact with test
samples, analytes, detection molecules or target molecules may be
used as a substrate. For example glass, quartz and plastic are used
as substrate in some embodiments. Substrates can be organic or
inorganic.
[0059] Any noble metal may be deposited. In various embodiments,
the film 220 of metal nanoparticles is one or more silver island
films (SIFs) deposited as described below or films of one or more
other metal nanoparticles such as gold, copper and aluminum.
[0060] Any method may be used to deposit the film of metal
nanoparticles 220. For example, the metal nanoparticles may be
deposited using a wet chemical deposition method to coat the
substrate 210 with the SIFs. In other embodiments, other methods
are used to deposit the metal nanoparticles, such as thermal vapor
deposition or deposition by sputtering, or patterning using
electron beam lithography. In some embodiments, an uneven film is
converted to more discrete particles, e.g., by annealing as
described in more detail below.
[0061] Any distribution of nanoparticle sizes may be deposited. In
a preferred embodiment, the nanoparticle sizes include a large
number in a size range that is small compared to the wavelengths of
fluorescent emissions from a particular fluorophore or a particular
set of one or more fluorophores to be used with the substrate. In
an example embodiment, the nanoparticles are deposited in a dense
configuration to reduce the area of voids where MEF does not occur.
The void dimensions are preferably small compared to a maximum
distance for effective MEF, such as 50 nm or less. In embodiments
that use a minor, as described in more detail below, it is
desirable that some incident light be allowed to reach the mirror
and be reflected. In such embodiments the nanoparticles are
preferably formed to have an optical densityin a range from about
0.2 to about 1.0, preferably about 1.0 (e.g., allow at least 10% of
the incident light to pass into the layer below the
nanoparticles.
[0062] Any molecule may be deposited in the bioactive molecule
layer 230. The properties of the functionalized substrate are
affected by the bioactive molecule deposited in layer 230. The
molecule should include a functional group to affix the molecule to
the substrate or metal film, such as a ligand to affix the molecule
to a metal nanoparticle. The molecule should also be able to bind
to a particular analyte of interest. Such a molecule is also called
a target molecule for an assay for the analyte. In illustrated
embodiments, all the molecules deposited in the layer 230 are
substantively identical. In other embodiments, functionalized
substrates are designed for multiple analytes and multiple
populations of different target molecules are used in the same well
for corresponding different analytes. Binding events of the
different analytes would be marked by fluorophores in corresponding
different detection molecules emitting at different optical
wavelengths. In various embodiments, target molecules that are
deposited in the layer 230 include, but are not limited to a
polymer, a ligand, an antigen, an antibody, a protein, a peptide,
DNA, RNA, or an oligonucleotide.
[0063] The functionalized substrate may be designed for any analyte
to bind to an appropriately chosen target molecule. In various
embodiments, the analyte includes, but is not limited to, a
polymer, a ligand, an antigen, an antibody, a protein, a peptide,
DNA, RNA, any form of RNA, an oligonucleotide, a virus, a bacterium
or a cell.
[0064] Region A 202 in a well 212 of the functionalized substrate
200 is indicated in FIG. 2A as an example portion of the substrate
210, film 220 and layer 230, e.g., as depicted in FIG. 1 and FIG.
4.
[0065] FIG. 3 is a block diagram that illustrates an example
fluorescence measurement apparatus suitable for sensing or imaging
fluorescence such as for an assay upon which an embodiment of the
invention may be implemented. The apparatus 300 is called a
epifluorescence measurement apparatus and is suitable for MEF
affinity assays. Apparatus 300 includes light source 310, such as a
light emitting diode (LED), lens 312, filters 330a and 330b, Epi
cube 340 and detector 350, such as a photo-multiplier tube (PMT) or
a charge-coupled device (CCD). In some embodiments a scanning
coupler 314 is included to obtain imagery data, e.g., in a FLIM
device. In other embodiments, a single measure is made of the well;
and, scanning coupler 314 is replaced with a simple coupler, such
as another lens. A computer controller 380, such as the computer
depicted in FIG. 23 or the chipset depicted in FIG. 24, is included
to control the operation of light source 310, scanning coupler 314,
if any, and to collect measurements from detector 350. The MEF
analysis module 382 performs one or more of the processes for
analyzing the data from a plasmonic substrate, such as determining
calibration curves and converting intensities and lifetimes to
concentrations of one or more analytes, as described in more detail
below.
[0066] Although an object of measurement 390 is depicted in FIG. 3,
the object 390 is not part of apparatus 300, but is operated upon
by apparatus 300. In some embodiments, the object 390 is a product
formed during an assay described below. During the experimental
measurements described below, object 390 is prepared in accordance
with one of the assays. Also depicted in FIG. 3 are light beams
produced during operation of the apparatus 300, including light
source light 360, filtered excitation light 362, solution emitted
light 372 and substrate emitted light 374.
[0067] Light from LED 310 is collimated with lens 312a, directed
through excitation bandpass filter 330a, dichroic splitter in Epi
cube 340, and scanning optical coupler 314 (such as a rotating
polygonal minor) to scan the object 390. The fluorescent light
emitted from the object 390 is passed through coupler 314, Epi cube
340, and emission filter 330b, and is collected at detector
350.
[0068] Lifetimes were determined, e.g., in module 382, using one-
or two-exponential models fit to the observations. These models are
represented by Equation 1.
I(t)=.SIGMA..sub.i.alpha.iexp(-t/.tau.i) (1)
where I is intensity at time t, i indicates an ith component of
several exponential components, exp is the exponential function in
which the base e is raised to the value of the argument inside
parentheses, .alpha.i is amplitude of the ith component and .tau.i
is the lifetime of the ith component at which time the component
falls to 1/e of its value at time t=0. The number of components
used is increased until a good fit is obtained for the data.
Amplitude weighted lifetimes <.tau.> are defined by Equation
2a.
<.tau.>=.SIGMA..sub.i.alpha.i*.tau.i (2a)
Intensity weighted lifetimes, .tau..sub.M, are defined by Equation
2b.
.tau..sub.M=.SIGMA..sub.ifi*.tau.i (2b)
Where fi is the fractional intensity defined by Equation 2c
fi=.alpha.i*.tau.i/.SIGMA..sub.i.alpha.i*.tau.i (2c)
[0069] The spectroscopic properties of various streptavidin-dye
conjugates in buffer solution are summarized in Table 1. Dye to
protein ratio (D/P) varied from about 1.0 to 3.9. Higher D/P ratios
than 4 usually lead to the self quenching and depolarization
effects. The spectral range of these fluorophores span a wide range
of wavelengths from about 495 nm to about 675 nm. The extinction
coefficient E is a physical property of the conjugate in solution,
is expressed in units of inverse Mole centimeters (M.sup.-1
cm.sup.-1, where 1 cm=10.sup.-2 meters) and is akin to an optical
cross section for interactions with an incident beam. The
wavelengths of maximum absorption (L.sub.s) and the wavelength of
maximum emission (.lamda..sub.EMs) are given in nanometers.
Intensity weighted lifetime and amplitude weighted lifetime are
given in nanoseconds (ns, 1 ns=10.sup.-9 seconds).
TABLE-US-00002 TABLE 1 Spectral properties of streptavidin-daye
conjugates in solution Intensity Decays Dye D/P .epsilon.
.lamda..sub.ABS/.lamda..sub.EMS .tau..sub.i (ns) .alpha..sub.i
f.sub.i <.tau.> .tau..sub.M Fluorescein-Bt N/A 68,000 494/518
4.11 1.0 1.0 4.11 4.11 AlexaFluor 488 3.9 71,000 495/519 3.08
0.4778 0.7382 1.759 2.418 0.55 0.5222 0.2618 DY495 1.0 70,000
495/520 3.91 0.4519 0.7991 2.211 3.287 0.81 0.5481 0.2009
AlexaFluor 532 4.1 81,000 529/551 2.60 0.1530 0.4968 0.804 1.533
0.48 0.8470 0.5032 DY547 1.0 150,000 553/572 3.76 0.0159 0.1926
0.316 0.934 0.26 0.9841 0.8074 Cy 3 1.7 150,000 558/578 1.23 0.3145
0.6684 0.579 0.915 0.28 0.6855 0.3316 AlexaFluor 635 1.5 140,000
632/647 4.88 0.3977 0.4681 3.995 4.037 3.41 0.6023 0.5139
AlexaFluor 647 2.7 239,000 653/669 2.11 0.6260 0.4447 1.485 1.183
0.44 0.3740 0.5553 Cy 5 0.9 250,000 657/678 1.86 1.0 1.0 1.86 1.86
AlexaFluor 680 2.8 184,000 679/675 1.98 1.0 1.0 1.98 1.98
[0070] Although processes, equipment, and data structures are
depicted in FIG. 3 as integral blocks in a particular arrangement
for purposes of illustration, in other embodiments one or more
processes or data structures, or portions thereof, are arranged in
a different manner, on the same or different hosts, in one or more
databases, or are omitted, or one or more different processes or
data structures are included on the same or different hosts.
[0071] FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D are block diagrams
that illustrate an example reference functionalized substrate
without a plasmonic substrate and other functionalized substrates
with two example plasmonic substrates, respectively, according to
multiple embodiments. FIG. 4A is a block diagram that illustrates
an example sandwich assay without a plasmonic substrate. A
plasmonic substrate is one that includes metal nanoparticles that
produce plasmons in response to excitation or emission light used
in a fluorescent assay. In this example a glass substrate 410
supports a layer 440 of fixed bioactive target molecules that have
affinity for an analyte. Deposited next is a layer 450 of the
detection molecule with fluorophores that have bound to any analyte
that binds to the target molecules in layer 440. The fluorophores
in this layer fluoresce in the response to excitation light with a
frequency of occurrence (and thus measured intensity) that depends
on the number of fluorophores bound to the analytes that are bound
to the layer of target molecules. This frequency of occurrence is
related to the concentration of the analyte in the sample fluid
which has since been washed away. The fluorescent output of each
fluorophore is indicated by a flash 471. Above the layer 450 is a
layer 460 of a covering solution, such as a buffer applied during
the last rinse.
[0072] FIG. 4B is a block diagram that illustrates an example
sandwich assay with a plasmonic substrate. Layers 410, 440, 450 and
460 are as described above. Layer 420 of metal nanoparticles on
glass substrate 410 produces a plasmonic substrate 431. The
proximity of the plasmonic substrate to the fluorophores in layer
450 of the bound detection molecules causes an increase of the
fluorescent emissions from each fluorophore as indicated by the
larger flashes 472. In general, the influence of plasmons induced
in plasmonic substrate 431 on the intensity of emission from the
fluorophores in layer 440 depends on the distance from layer 450 to
layer 420 being on the order of about 3 to 50 nanometers. This
distance can be assured by a proper choice of sizes and binding
sites for the target molecules in layer 440 and the detection
molecules in layer 450 and the added distance of the analyte
sandwiched on the interface of those two layers. The combination of
a plasmonic substrate, such as substrate 431, and the layer 440 of
fixed target molecules is called the functionalized substrate
hereinafter. The fluorescent output of each fluorophore is
indicated by a flash 472, that is substantially greater than the
output 471 without a plasmonic substrate.
[0073] FIG. 4C is a block diagram that illustrates an example
sandwich assay with a superior plasmonic substrate 432, according
to some example embodiments. The plasmonic substrate 432 includes,
between the glass 410 and a layer of metal nanoparticles, a minor,
such as silver (Ag) mirror 424 and a dielectric layer, e.g.,
silicon oxide (SiOx) layer 423. In some embodiments, the dielectric
layer may comprise silicon monoxide or silicon dioxide. In an
experimental embodiment illustrated, the layer of metal
nanoparticles is a layer 422 of silver film formed of silver
nanoparticles, as described in more detail below. In some
embodiments, the layer of nanoparticles includes any metal
including, for example, silver, gold, or aluminum, as particles or
a film with undulating surface. In various embodiments, the
metallic mirror layer is constructed of any metal, such as silver,
gold, or aluminum. In the illustrated embodiment, the Ag mirror is
fixed to the glass substrate 410 by an adhesive layer, e.g., layer
425 of Chromium (Cr) film. In other embodiments, the adhesive layer
may consist of any non-metallic material that will prevent slipping
between the base layer and the metallic mirror layer, such as
titanium oxide or silicon nitride. In the experimental embodiment,
the layer 460 of supernatant fluid is a layer 462 of a buffer
solution, such as phosphate buffer saline (PBS). In this
embodiment, the detection molecule layer 450 is a layer 452 of
dye-streptavidin conjugate (Dye-SA) bound to the analyte that binds
to the target molecule layer (where the dye is one of the
fluorophores in Table 1). Also, in this embodiment, the fixed
target molecule layer 440 is a layer 442 of biotinylated bovine
serum albumin (BSA-Bt). The fluorescent output of each fluorophore
is indicated by a flash 473 that is substantially greater than the
output 472 from the plasmonic substrate of FIG. 4B or the output
471 without a plasmonic substrate. FIG. 4D is a block diagram that
illustrates an example perspective view of a small portion of a
plasmonic substrate, according to an embodiment. The layers 422,
423, 424 and 410 are as described above for FIG. 4C. The enhanced
far field fluorescent emissions are represented by the yellow
arrows that correspond to flashes 473.
[0074] For the substrates with Ag nanoparticles on the glass as
depicted in FIG. 4B, it was found that excitation and observation
at the other side of the glass (opposite to the protein-fluorophore
layers) resulted in fluorescence signals comparable to those for
direct excitation/observation conditions. This implies that for
direct excitation/emission configuration, a significant amount of
fluorescence is coupled into a glass substrate and radiated in the
opposite direction to the detector. This is because the under layer
of glass has a higher permittivity than the aqueous solution above
the silver nanostructures, and highly efficient coupling of
fluorescence occus into the glass substrate 410. Therefore,
inventors determined to include a proper mirror and layer of
dielectric to redirect the scattered light toward the observation
path. The scattered light trapped in dielectric layer is reflected
by the mirror which efficiently increases the excitation and
redirect fluorescence into the observation path. The process of
reflections within a dielectric layer will occur multiple times
since the outer layer of silver nanostructures is
semitransparent.
[0075] This configuration of layers was found to have a
surprisingly large enhancement of fluorescent far field emission
intensity (up to about 200-fold), indicated by the much larger
flashes 473. Associated with this surprising enhancement is a
commensurate surprising decrease in fluorescence lifetime.
Decreases in lifetime associated with increases in emission
intensity are characteristic of plasmon-fluorophore
interactions.
[0076] The inventors recognized that such increases in intensity
and decreases in lifetime as depicted in FIG. 4B and FIG. 4C made
it easy to distinguish fluorophores in solution from those bound in
the vicinity of the layer of metal nanoparticles, and direct
measurements are enabled in some assay embodiments so that there is
no need to rinse either the sample with analyte or the reagent with
detection molecules from the covering solution above the
functionalized substrate.
[0077] This ability to provide direct measurements without rinsing
away a sample fluid has great advantages for detection and analysis
of secretions, such as cytokines, from cells.
[0078] FIG. 5 is a block diagram that illustrates an example
measurement of quantity of analytes from a single cell, according
to an embodiment. A plasmonic substrate 530 (such as plasmonic
substrate 431 of FIG. 4B or plasmonic substrate 432 of FIG. 4C)
supports a layer 532 of fixed target molecules. A cell 590 secretes
analytes 570 (such as one or more different cytokines). These
analytes bind to the detecting molecules 572, either in solution or
bound to the fixed target molecules 532. The unbound detection
molecules fluoresce with un-enhanced intensity and non-shortened
lifetimes indicated by small flashes 471, while, at least after a
time (indicated by the horizontal arrow pointing to the right), the
detection molecules bind to the target molecules via secreted
protein (analyte) and fluoresce with enhanced intensity and
shortened lifetimes indicated by large flashes 473. Upon binding a
spot with increased intensity and decreased lifetime is formed. The
distinction can be observed even without rinsing the reagent with
detection molecules 572, or the sample with cell 590, from the
covering solution. With the surprisingly enhanced emissions of 473,
even low levels of an analyte can be detected, such as is the case
with cytokines secreted from a single cell. The change in enhanced
emission or lifetimes over time can be monitored as the cell
continues to live in the sample during the measurements. This
capability is not believed to be present in the prior art
approaches.
[0079] FIG. 6 is a flow chart that illustrates an example method to
directly assay the quantity of an analyte in a sample without
removing the sample, according to an embodiment. Although steps are
depicted in FIG. 6 as integral steps in a particular order for
purposes of illustration, in other embodiments, one or more steps,
or portions thereof, are performed in a different order, or
overlapping in time, in series or in parallel, or are omitted, or
one or more additional steps are added, or the method is changed in
some combination of ways. For example, in some embodiments, one or
more rinsing steps are added.
[0080] In step 601, a functionalized substrate is provided. In an
illustrated embodiment, the functionalized substrate includes a
plasmonic substrate with a layer of metal nanoparticles that are
smaller than wavelengths emitted from a particular set of one or
more fluorophores to be used in an assay. In this embodiment, the
functionalized substrate also includes a layer of one or more
populations of substantively identical bioactive target molecules
that bind to a particular analyte of interest for corresponding one
or more analytes of interest. In some embodiments, the target
molecules for different analytes are in separate wells. In other
embodiments, target molecules for two or more analytes are included
in the same well, either intermixed or segregated.
[0081] The functionalized substrate can be provided in any manner.
For example, in some embodiments, the substrate is obtained (e.g.,
from a commercial supplier) with both the metal nanoparticles and
layer of bioactive molecule. In some embodiments, the substrate is
obtained with the metal nanoparticles already deposited but without
the bioactive layer, and the bioactive layer is deposited during
step 605. In some of these embodiments the bioactive molecule is
supplied and shipped in a separate container (e.g., to preserve its
efficacy) as part of an assay kit, and deposited during step 605 to
form the functionalized substrate when desired for use. In some
other embodiments, the substrate is obtained with neither the metal
nanoparticles nor the bioactive layer. In such embodiments, the
metal nanoparticles are deposited during step 603 to form a
plasmonic substrate, and the bioactive molecule layer is deposited
during step 605. In some embodiments, step 603 includes depositing
a reflective metal layer configured as a minor, and depositing on
the mirror a dielectric material, and depositing the metal
nanoparticles on top of the dielectric layer.
[0082] Any metal may be used in the metal nanoparticles in various
embodiments, such as gold, silver, copper and aluminum, or some
combination. Any molecule may serve as the target molecule in the
bioactive layer, such as a polymer, a ligand, an antigen, an
antibody, a protein, a peptide, DNA, RNA, or an
oligonucleotide.
[0083] In step 611, a reagent is provided, typically in solution.
The solution of reagent includes a known quantity of a detection
molecule comprising a probe and a fluorophore. The probe is
selected to assay for the particular analyte. The probe is labeled
with a particular fluorophore from the particular set of
fluorophores with emission wavelengths suitable for plasmon light
interactions. The reagent can be provided in any manner. For
example, in some embodiments, the reagent is obtained from a
commercial supplier. In some embodiments, the reagent is provided
in an assay kit that also includes the plasmonic substrate and the
bioactive molecule in a separate container. In some embodiments the
reagent is prepared locally by a user of the assay. In some
embodiments, the reagent includes known concentrations of each of
several different detection molecules, each with corresponding
different fluorophores and each with affinities for corresponding
different analytes, e.g., different cytokines secreted from a
single cell.
[0084] Any molecule may be included as the probe in the detection
molecule, such as a polymer, a ligand, an antigen, an antibody, a
protein, an oligomer, a protein, a peptide, DNA, RNA or an
oligonucleotide. Any fluorophore may be included in the detection
molecule, such as fluoresceins, eosin, coumarines, rhodamines,
cyanines, benzophenoxazines, phycobiliproteins or fluorescent
proteins.
[0085] In step 613 a test sample is obtained with a quantity of a
particular one or more analytes to be determined by the assay.
During a calibration phase used in some embodiments, step 613
includes providing a control sample with known quantities of the
one or more particular analytes. For assays that are previously
developed, with a known calibration curve, a control sample is not
used during step 613. The quantity (such as the presence or
concentration) of each of the one or more analytes in the test
sample is determined during step 623, described below. Any material
may serve as the one of the one or more analytes, such as a
polymer, a ligand, an antigen, an antibody, a protein, a cytokine,
a peptide, DNA, RNA, oligonucleotide, a virus, bacterium, or a cell
from a patient.
[0086] In step 615, the functionalized substrate is contacted with
the test sample and the reagent for sufficient time to produce
binding of the one or more different detection molecules to the one
or more different analytes or to produce binding of the one or more
different analytes to the one or more different fixed bioactive
target molecules. To monitor temporal progression of a
cell-oriented process, steady state conditions do not need to be
reached. Unlike previous sandwich assays, the functionalized
substrate need not be brought into contact with the test sample
first and allowed to remain in contact for sufficient time to allow
the analyte to bind to the target molecules fixed to the substrate
in amounts that are proportional to the amount of analyte in the
test sample. The functionalized substrate with bound analyte does
not then need to be washed to remove excess analyte. The
functionalized substrate with bound analyte is not then contacted
with the solution of the reagent in a separate step and then
maintained for sufficient time to allow the detection molecule to
bind to the analyte fixed by the target molecule to the
functionalized substrate. The functionalized substrate does not
need to be rinsed again to remove the excess detection
molecules.
[0087] In step 617, the substrate and covering solution resulting
from step 615 are exposed to excitation electromagnetic waves, such
as light, that excites fluorescence in the one or more particular
fluorophores corresponding to the different analytes.
[0088] In step 619 the relative intensity of emission
electromagnetic waves is measured at the emission wavelength of the
fluorophore corresponding to each of the one or more analytes. In
some embodiments, the measurement is made relative to a reference,
such as a control well with no analyte or a control well with no
functionalized substrate. In some embodiments, the measurement is
made relative to an area of minimum intensity. In the illustrated
embodiment, step 619 overlaps in time step 617, as the substrate
and covering solution are excited and fluoresce measured at the
same or overlapping times. In some embodiments that include step
621, step 619 is omitted.
[0089] In step 621 the relative lifetime of emission
electromagnetic waves is measured at the emission wavelength of the
fluorophore corresponding to each of the one or more analytes. In
some embodiments, the measurement is made relative to a reference,
such as a control well with no analyte or a control well with no
functionalized substrate. In some embodiments, the measurement is
made relative to an area of maximum lifetime, since plasmons tend
to reduce the lifetime of fluorescent emissions. Any method may be
used to measure lifetime, such as measuring time-dependent emission
intensity and fitting to a one or two decay coefficients as
described above with reference to Equation 1. In some embodiments,
lifetime is measured by the phase difference between amplitude
modulated excitation and emitted waveforms, with increasing
lifetimes causing larger phase differences. In the illustrated
embodiment, step 621 overlaps in time step 617, as the substrate
and covering solution are excited and fluoresce measured at the
same or overlapping times. In some embodiments that include step
619, step 621 is omitted.
[0090] The lifetime measurements provide valuable information about
the mechanism of the enhancement. For example, if the intensity is
enhanced but the lifetime is not changed, this means that the
mechanism of intensity enhancement is due to enhanced excitation
intensity or an increase in the fluorophore concentration. However,
if the fluorescence enhancement is accompanied by a lifetime
decrease, the effect of the surface plasmons on the decay rates of
the fluorophore needs to be considered. In some embodiments, phase
modulation fluorometry is used to acquire data on lifetime changes
at the same time as steady-state intensity measurements.
[0091] In step 623, a particular quantity of analyte bound to one
or more areas on the functionalized substrate is associated with
the measured value of relative intensity or lifetime or both.
During a calibration phase, the known quantity of analyte in the
control sample is associated with the measured values to add points
to the calibration curves.
[0092] In step 625, one or more analyses of the sample are
performed based on the quantities of the bound analytes. For
example, one or more functions of an immune system cell are
determined by a profile of cytokines secreted during measurement.
As another example, a rate of secretion of the analyte by cell is
determined based on a difference with a prior or subsequent
measurement. In some embodiments, step 625 includes exposing the
sample to one or more stimulants, e.g., to induce an immune
reaction in a sample that includes one or more cells of an immune
system.
[0093] In step 627, it is determined whether to make a measurement
of the same sample at another time. This is only possible because
the sample and reagent do not need to be rinsed off to make the
measurement. If so, then control passes back to step 617 to expose
the sample again to excitation electromagnetic waves. If not, then
another sample, if any, is measured on another substrate, e.g., on
another well of the same glass substrate or on other slide
altogether, e.g., by returning to step 601 or step 613. In some
embodiments, the next measurement is with another known quantity of
analyte in another control sample to produce another point for the
calibration curve. In a post calibration operational phase, a
quantity on the established calibration curve associated with the
measured intensity or lifetime, or both, is determined to be the
quantity of the analyte in the test sample. The quantity indicates,
for example, the presence, absence or concentration of the
analyte.
[0094] As described in more detail below, the inventors developed
planar plasmonic substrates that provide fluorescence amplification
of more than 200-fold and demonstrated cytokine assay sensitivities
in the range of 10 pg/ml and below.
[0095] Further, the inventors demonstrated that the developed
plasmonic substrates are robust and applicable to cellular studies.
Because this approach is similar to ELISPOT, these MEF-based assays
for single cell analysis are called MEFspot. The MEF-based approach
provides an opportunity for platform technology applicable to a
broad range of immunoassays including single cell assays. The
MEF-based technology described has attributes of ELISA (sensitivity
and application to liquid samples), ELISPOT (sensitivity and
applications to single cells), and flow cytometry (quantitation of
production of cytokines by single cells).
[0096] In addition there are several advantages. There is no
requirement for specific assay reagents; standard kits for sandwich
type assay are used in various embodiments. There is no requirement
for specific fluorescent probes; every fluorophore undergoes MEF.
There is no requirement for special detection devices; all
fluorometric readers are used in various embodiments; microscopy is
used for imaging cell populations. There is no requirement for new
special procedures. Procedures are simplified compared to ELISA and
ELISPOT. The techniques are amendable for high-throughput and
printable protein arrays. The techniques are easy to customize for
particular assays, are easy to use as a one-step assay, and are
cost effective because they are effective with small samples, small
cell numbers, and the plasmonic substrates can be mass
produced.
[0097] Advantages of the high contrast generated by minute amount
of bound probe in MEFspot results in new capabilities for cellular
studies not available with other techniques, including: real-time
monitoring of protein secretion; direct quantification of
secretion; dual modality for secretion verification using both
intensity and lifetime; multiplexing and simultaneous detection of
cell phenotype and function; and, monitoring simultaneously cell
viability and cell function.
[0098] It is anticipated that MEFspot will be regarded as a highly
innovative detection platform for immunoassays and cellular
analysis of heterogeneous cell populations which will have high
impact in cancer research, immunology, diagnostics, and therapeutic
development, among others.
[0099] Some embodiments of the invention are directed to an MEFspot
assay kit. In these embodiments, the user is a recipient of the
kit. In one embodiment, this kit includes a plasmonic substrate and
one or more containers of target molecules in solution to
functionalize the substrate for assays for one or more
corresponding analytes. In some embodiments, the kit also includes
one or more containers of detection molecules for the one or more
corresponding analytes. In some embodiments, the kit includes
containers of unlabeled biomolecules to serve as probes for
detection molecules for the one or more corresponding analytes, and
containers for one or more fluorophores. Recall that a detection
molecule includes a probe and a fluorophore. The fluorophores are
selected from a particular set for which emission wavelengths are
long compared to the nanoparticle sizes on the substrate. The user
labels the biomolecules with a fluorophore chosen from an included
container to produce a reagent of detection molecules. In a
preferred embodiment, the probe molecules are already labeled with
the selected fluorophore and provided in a container as a reagent
of detection molecules. In some embodiments, one or more control
samples with known quantities of analyte are also included to be
used to generate calibration curves. In some embodiments, printed
media or a computer-readable medium is included with data that
indicates a calibration curve and software to compute the various
analyses that can be performed with the data, such as phasor
diagrams described below.
[0100] The MEFspot assay kit provides the recipient with materials
to perform a MEFspot assay on the recipient's own one or more test
samples according to one or more embodiments of method 800.
2. Example Embodiments
[0101] Here are described planar plasmonic substrates that provide
fluorescence amplification of about 200-fold and demonstrate
cytokine assay sensitivities in the range of 10 pg/ml and
below.
2.1 Plasmonic Substrates
[0102] In some embodiments, silicon monoxide and silver wire
(99.999%) were obtained from SIGMA-ALDRICH.TM. of St. Louis, Mo.
Streptavidin (SA) or avidin (Av) conjugated dyes, Alexa Fluor 350
(AF350-Av), Alexa Fluor 488 (AF488-SA), and Alexa Fluor 647
(AF647-SA) were obtained from INVITROGEN.TM. of Carlsbad, Calif.).
Phosphate buffer saline (PBS) pH 7.4 and biotinylated bovine serum
albumin (BSA-Bt) were from SIGMA-ALDRICH.TM.. Ultrapure water
(>18.0 M.OMEGA.) purified using a Millipore Milli-Q gradient
system was used in preparation of buffers and aqueous
solutions.
[0103] For fabrication of multilayered plasmonic substrates, in
some embodiments, glass microscope slides were purchased from
VWR.TM. of Radnor, Pa. Glass slides were cleaned with "piranha
solution" (35% H2O2/H2SO4, 1:3) overnight, rinsed with distilled
deionized water, and dried with nitrogen before thermal vacuum
deposition steps. Metallic and dielectric layers were deposited by
thermal evaporation (Edward, model 306) or magnetron sputtering
(AJA model ATC 1800-V). For thermal deposition, chromium (adhesion
layer) and silver (mirror and outer layer) were evaporated from
tungsten boats at 2.times.10.sup.-7 Torr and silicon monoxide at
5.times.10.sup.-6 Torr with a deposition rate of .about.1.0
nm/minute. In some embodiments, after coating with silicon monoxide
(or silicon dioxide), slides were silanized by immersion in a water
solution of 1% of aminopropyl trimethoxysilane (APS), for 30 mM.
The silanized slides were dried in air and used for deposition of a
final thin layer of Ag followed by thermal annealing in air at
various temperatures and various annealing times.
[0104] For evaluation of fluorescence enhancements, the surfaces of
annealed and non-annealed slides were covered with a self-adhesive
silicone/rubber of thickness of 2 millimeter (m, 1 mm=10.sup.-3
meters) with wells of 2.5 mm diameter. First, the BSA-Bt solution
(100 micograms per milliliter, .mu.g/ml, 1 mg=10.sup.-6 grams) in
sodium phosphate buffer (50 milliMolar, mM, 1 mM=10.sup.-3 Molar,
pH of 7.2) was added into the wells (10 .mu.l, 1 .mu.l=10.sup.-6
liters) and incubated for 1 hour. This step facilitated a monolayer
of BSA-Bt that provided the means for immobilization of
streptavidin-dye conjugates. The same procedure was used for
preparation of control samples using bare glass slides. After
incubation with dye-streptavidin conjugates (25 .mu.g/ml), the
wells were washed with PBS buffer to remove unbound dye
streptavidin conjugates. Finally, the wells were filled with PBS
and covered with a microscope coverslip for spectroscopic
measurements. The schemes of multilayer substrates with immobilized
dye-streptavidin conjugates are shown in FIG. 4C and FIG. 4D.
[0105] In some embodiments, absorption spectra were acquired with a
Hewlett-Packard 8453 spectrophotometer. For baseline corrections,
bare glass substrate and SiOx coated glass slides were used.
Steady-state intensities were measured on the multilayer substrates
and compared with the signal of the respective samples on bare
glass. Fluorescence enhancement was determined as the intensity
ratio of the fluorescence signal measured on the multilayer
substrate divided by the signal in respective reference sample on
bare glass using identical experimental conditions. Fluorescence
from surfaces was measured with epi-fluorescence configuration
(e.g., see FIG. 3 with lens instead of scanning coupler 314) using
a fluorescence microscope (Axiovert 135TV, Zeiss) with a 10.times.,
NA 0.30 objective (UPlanFl, Olympus). The excitations were provided
using ultraviolet (UV) lighe emitting diode (LED) (Nichia NSHU590E)
with a peak wavelength at 374 nm, blue LED (Nichia NSPB500S) with a
peak wavelength at 467 nm, and red LED (Nichia NSPR510CS) at 625 nm
and emission observed at band-pass filters of 460/50 nm (AF350-Av),
535/50 nm (AF488-SA), and long pass filter above 655 nm (AF647-SA).
Time-resolved data were measured using a phase-modulation
fluorometer (K2 from ISS, Champagne, Ill.). The LEDs were modulated
by applying a RF driving signal from a Marconi model 2022A
frequency synthesizer (from Marconi Instruments, Allendale, N.J.)
to the LED.
[0106] Scanning electron microscopy (SEM) images were collected
with a Hitachi SU-70 SEM instrument, and surface morphologies were
studied using an atomic force microscope (AFM), model D3000 (from
Digital Instruments, Inc.).
[0107] A set of silver films with nominal thicknesses 9, 15, 20,
23, 30, and 36 nm were prepared by thermal evaporation of silver
(Ag) onto silanized SiOx coated microscope slides. Part of the
slide from each set was annealed for 3 hours at 230.degree. C. in
air. SEM images and absorption spectra of annealed and non-annealed
films were obtained (not shown). In some embodiments, the
measurements of absorption spectra were performed for surfaces
wetted with phosphate buffer, pH 7.2
[0108] In some embodiments, the layer of metal nanoparticles for
the plasmonic substrate obtains improved performance by annealing.
FIG. 7A, FIG. 7B and FIG. 7C are graphs that illustrates example
effects of annealing on shape and optical absorption of
nanoparticles in a plasmonic substrate, according to an embodiment.
FIG. 7A is a graph 710 that illustrates example absorbance spectra
of annealed and non-annealed Ag layers of 20 nm effective
thicknesses on the glass substrate, which were fabricated
simultaneously with Ag mirrored substrates coated with dielectric
layers. The horizontal axis 712 is optical wavelength and the
vertical axis 714 indicates absorbance in units of optical desnity.
Graph 710 shows the respective absorption spectra of annealed
(trace 716a) and non-annealed (trace 716b) substrates and three
excitation wavelengths (arrows) and three spectral windows
(horizontal bars) used for evaluation of fluorescence enhancements.
The selected spectral windows overlap with the plasmon spectrum
over a broad range of wavelengths representative of the fluorescent
dyes commonly used in biotechnology applications. AFM images 700a
for annealed and 700b for non-annealed show that the annealing
process results in substantial changes in the surface morphology,
increasing both lateral and axial dimensions of Ag nanostructures.
FIG. 7B is a graph 720 that illustrates an example particle height
profile across the annealed image 700a. The horizontal axis 722 is
distance in nanometers and the vertical axis is height of particle
in nanometers. FIG. 7C is a graph 730 that illustrates an example
particle height profile across the non-annealed image 700b. The
height profiles indicate that smaller silver nanostructures are
formed during the vacuum deposition, with an average height close
to the effective deposition thickness of 20 nm. After annealing,
the height of the particles increased up to about 60 nm and the
lateral size also increased up to about 150 nm.
[0109] Effects of the above fabricated Ag nanostructures on the
fluorescence were determined in on embodiment using three
fluorophores (AF350, AF488, and AF647). The selected fluorophores
have distinct spectral ranges representing coumarins (UV-blue),
fluoresceins (blue-green), and cyanines (red), respectively. The
selected dye-strept (avidin) conjugates display similar quantum
yields: 0.55 (AF350-Av), 0.42 (AF488-SA), and 0.33 (AF647-SA).
Similar quantum yields of fluorophores allow more proper
characterization of the plasmonic effects of Ag nanostructures on
fluorescence enhancement over the broad spectral range. It is known
that low quantum yield fluorophores undergo larger fluorescence
enhancements compared with that of high quantum yield. Additional
advantages of using Alexa Fluors with plasmonic nanostructures are:
their better photostabilities; and less self-quenching when labeled
with proteins, compared with conventional dyes. The avidin and
streptavidin conjugated dyes were immobilized on substrates that
were precoated with BSA-Bt. The binding interaction between
streptavidin and biotin is very strong and results in a stable
monolayer of dye-streptavidin over the BSA. The layer of BSA-Bt
serves also as a separation layer between the fluorophores and the
silver surface. The average distance between fluorophores (bound to
SA) and the surface is about 6 to about 7 nm. This separation
prevents fluorescence quenching when the dye is in direct contact
with the metallic surface or in close proximity to the surface
where the quenching effects are more dominant over the enhancement
effects. All investigated substrates, including control bare glass
slides, were treated with the same protein concentrations to
facilitate a direct comparison of fluorescence signals
[0110] Because the annealing process has a significant effect on
the Ag film morphology and on the overall fluorescence
enhancements, the effect of annealing temperature and annealing
time on the performance of Ag film-based substrates was
investigated. For this study, silver films with a thickness of 30
nm were deposited on the glass substrates having a 25 nm SiOx under
layer and annealed at various temperatures for a fixed time (2
hours). A Ag thickness of 30 nm was selected to possibly maximize
the annealing effect on the fluorescence enhancement. Note that
this Ag thickness is not optimal for enhancement. The second set of
substrates was annealed for different times at a fixed temperature
(180.degree. C.). The fluorescence enhancements for annealed and
non-annealed films showed that the optimal annealing temperature is
in a broad range from about 100 to 200.degree. C., and sufficient
annealing time is from about 5 minutes to about 60 minutes,
preferably about 60 minutes.
[0111] FIG. 8A and FIG. 8B are block diagrams that illustrate
example configurations in the vicinity of a substrate for which
optical fields are simulated to determine the effects of the
dielectric layer thickness on fluorescence enhancement, according
to an embodiment. FIG. 8A depicts a reference model with a layer
860 of water overlaying a layer 810 of glass. Incident light has an
electric field E.sub.Y0 that is polarized parallel to the interface
between the layers 810 and 860. The average excitation field
intensity and radiation power of randomly distributed dipoles were
computed for a 10 nm layer 880a above the interface. FIG. 8B
depicts a model of an advantageous plasmonic substrate. The model
plasmonic substrate includes glass layer 810 and water layer 860
but includes a silver (Ag) mirror layer 824 above the glass, a
silicon dioxide (SiO.sub.2) layer 823 above the mirror, and a
silver (Ag) nanoparticle layer 822 above the SiO.sub.2 layer 824.
The average excitation field intensity and radiation power of
randomly distributed dipoles were computed for a 10 nm layer 880b
above the interface of the layers 822 and 824 with the water 860.
The parameters of the plasmonic substrate model include Ag array of
semi-spheres with diameter of 60 nm and 40 nm edge-to-edge spacing,
Ag mirror with thickness of 48 nm, SiO.sub.2 layer with variable
thickness from 0 to 240 nm and water thickness of 500 nm.
[0112] Numerical calculations were based on the finite-element
method (FEM) using COMSOL Multiphysics version 3.5a from COMOL INC.
.TM. of Burlington, Mass. Two mechanisms of fluorescence
enhancement caused by the multi-layered substrates were considered:
enhancement in the excitation and enhancement in the emission
confined to the region where fluorescent probes are located. Thus
we performed two set of calculations: (1) calculating the effect of
coupling of excitation light into a multi-layered substrate via
average field intensity within a 10 nm layer above the surface and
(2) calculating the emission enhancement of randomly distributed
and randomly oriented dipoles (representing fluorophores) within a
10 nm layer above the Ag nanoparticles.
[0113] For experimental comparisons, in some embodiments, glass
slides (from VWR) were cleaned with "piranha solution" (35%
H.sub.2O.sub.2/H.sub.2SO.sub.4, 1:3) overnight, rinsed with
distilled deionized water and dried with nitrogen before performing
vacuum deposition steps. Metallic and dielectric layers were
deposited using magnetron sputtering (AJA Model ATC 1800-V from AJA
INTERNATIONAL INC. .TM. of Scituate, Mass.). First, a 1.5 nm
chromium layer was deposited on glass for adhesion of an Ag layer
of 200 nm thickness. After deposition of Ag film, a silicon dioxide
layer with thickness varying from 3 to 300 nm. A final thin layer
of Ag of about 13 nm was deposited followed by thermal annealing in
air at 180.degree. C. for 1 hour.
[0114] The surfaces of the substrates and reference glass slides
were coated with biotinylated bovine serum albumin (BSA-Bt) using
100 .mu.g/ml solution in phosphate buffer with an incubation time
of 1 hour. This step formed a monolayer of BSA-Bt that facilitated
the immobilization of streptavidin-dye conjugates (5 .mu.g/ml).
Streptavidin (SA) conjugated dyes are: Alexa Fluors from
INVITROGEN.TM. AF488-SA, AF647-SA, AF680-SA, and AF750-SA; and the
infra red dye IRD800-SA from LICOR.TM. of Lincoln, Nebr. Phosphate
buffer (PB) pH 7.4 and biotinylated bovine serum albumin (BSA-Bt)
were from SIGMA-ALDRICH.TM.. Ultrapure water (>18.0 M.OMEGA.)
(Millipore Milli-Q gradient system) was used in the preparation of
buffers and aqueous solutions.
[0115] Extinction spectra were measured with a Hewlett-Packard 8453
spectrophotometer from HEWLETT-PACKARD Palo Alto, Calif., relative
to the bare glass. Reflectance spectra were acquired with a Cary
100 Bio spectrophotometer from VARIAN MEDICAL SYSTEMS INC..TM. of
Palo Alto, Inc., equipped with an external specular reflection
attachment with fixed angle of incidence of 12 degrees. For
baseline correction, a reference aluminum mirror (reflectance
accessory) was used. Scanning Electron Microscopy (Hitachi SU-70
from HITACHI, LTD. Tokyo, Japan) was used for surface morphology
imaging. Fluorescence from surfaces was measured using an
epi-fluorescence microscope (Axiovert 135TV from ZEISS GMBH.TM. of
Jena, Germany, see FIG. 3) with 10.times., NA 0.30 objective
(UPlanFl from OLYMPUS CORPORATION of Tokyo, Japan). Excitation was
provided using either a blue LED (NSPB500S from NICHIA CORPORATION
of Tokushima, Japan) with peak wavelength at 470 nm or a red LED
(NSPR510CS from NICHIA CORPORATION) at 630 nm, and the emission was
observed through a band pass filter of 535/50 nm (AF488-SA) and
long pass filter above 655 nm (red dyes). An NIR reader Odyssey
from LICOR.TM. was used for NIR dyes at laser excitation of 680 and
780 nm. Steady-state intensities were measured on the multi-layer
substrates and compared to the signal of the respective samples on
bare glass. Fluorescence enhancement was determined as the
intensity ratio of the fluorescence signal measured from the
multi-layer substrate over the signal from the respective reference
sample on bare glass using identical experimental conditions.
Time-resolved data were measured using phase-modulation fluorometer
(K2 from ISS, Champagne, Ill.). The LEDs were modulated by applying
a RF driving signal from a Marconi Model 2022A frequency
synthesizer (from MARCONI INSTRUMENTS.TM., Allendale, N.J.) to the
LED.
[0116] FIG. 9A and FIG. 9B are graphs that illustrate the ability
of the simulation to match data, according to an embodiment. FIG.
9A is a graph 910 and image inset 900. The inset 900 presents an
example scanning electron micrograph (SEM) image of Ag
nanoparticles obtained after annealing an Ag film with thickness of
13 nm on silica coated glass. The Ag nanostructures can be
considered as a collection of nanoparticles heterogeneous in size
and shape. Using an imaged area of 2.times.2 .mu.m.sup.2, the
estimated filling factor is 24%, with about 11% of the area covered
with circular particles of average diameter of 81.+-.26 nm and 13%
with elongated particles of average size 77 nm.times.132 nm.
[0117] Graph 910 has a horizontal axis 912 that indicate optical
wavelength in nanometers, and a vertical axis 914 that indicates
extinction, a dimensionless quantity. Calculated extinction (E) is
determined from the calculated transmission (T) of incident light
using the formula E=log(1/T). The graph depicts example extinction
spectrum of Ag nanoparticles on silica coated glass (solid line
916b) and the numerically calculated spectrum for Ag array of
semispherical nanoparticles of 60 nm diameter and edge-to-edge
spacing of 40 nm (dashed line 916a). There is good qualitative
agreement.
[0118] FIG. 9B is a graph 920 that illustrates example absorption
and emission spectra ofselectedifluorophores. The horizontal axis
922 is optical wavelength in nanometers and the vertical axis 924
indicates normalized intensity in arbitrary units. Graph 920
depicts absorption spectra (thin lines 926a, 927a and 928a) and
emission spectra (thick lines 926b, 927b and 928b) of the AF488-SA
(1), AF647-SA (2), and AF750-SA (3) fluorophores, respectively. For
clarity of figure, spectra of other fluorophores are not included.
Arrows indicate the experimental excitation wavelengths within the
absorption peaks for some embodiments.
[0119] When the silica thickness is increased above the 75 nm in
some embodiments, the reflectance spectra are more complex,
displaying well-defined minima and maxima specific to the
particular silica thickness. Reflectance properties of the
substrates indicate that incident light can be highly reflected,
almost to 100%, as well as can be highly absorbed or transmitted
(low reflectance of about 5%). Measurements of UV/Visible/near
infrared (NIR) reflectance and extinction suggest a complex
interaction of light with plasmonic structures coupled to a
photonic cavity. Therefore, in order to better understand the
fluorescence enhancement and to gain insights on rational design of
the substrates, numerical calculations were used to determine the
effect of variation in silica thickness of multi layered substrates
on the excitation and emission enhancements for selected
wavelengths. An important factor is the interplay between Ag
surface plasmon resonance and resonance within a silica layer which
can be constructive as well as destructive as indicated by the
reflectance spectra.
[0120] The excitation enhancement was calculated as the ratio of
average electromagnetic power within the 10 nm volume above the
surface of the substrate to that of bare glass with the same silica
thicknesses. FIG. 10A, FIG. 10B and FIG. 10C are graphs 1010, 1020,
1040 that illustrate example enhancement of excitation light
intensity based on thickness of dielectric layer, according to
various embodiments. The horizontal axis 1002 indicates thickness
of the SiO2 layer. The vertical axes 1004, 1024, 1044,
respectively, indicate the excitation enhancement, which is
dimensionless. FIG. 10A is for a plasmonic substrate with minor,
dielectric layer and Ag nanoparticles. FIG. 10B is for a plasmonic
substrate with dielectric layer and Ag nanoparticles. FIG. 10B is
for a substrate with a mirror and dielectric layer abut no Ag
nanoparticles. On each graph there are five traces corresponding to
five excitation wavelengths. For the three graphs, respectively,
enhancements at 470 nm are shown on traces 1011, 1031, 1051;
enhancements at 530 nm are shown on traces 1012, 1032, 1052;
enhancements at 630 nm are shown on traces 1013, 1033, 1053;
enhancements at 680 nm are shown on traces 1014, 1034, 1054; and,
enhancements at 780 nm are shown on traces 1015, 1035, 1055. These
calculations indicate that the substrate with minor, silica cavity
and AgNP provides the most efficient excitation enhancements.
[0121] The substrate with silica layer thickness of approximately
25-60 nm facilitates similar enhancements across a broad range of
excitation wavelengths. Using greater silica thicknesses above 150
nm, one can also obtain high excitation enhancement (second peak)
which varies strongly with wavelength; therefore multiple
spectrally-distinct fluorophores may display very different
enhancements or even quenching. Such strong wavelength dependence
may be useful for construction of substrates for detection systems
where some wavelengths need to be enhanced and other suppressed.
Thus plasmonic substrates with thickness from about 20 nm to about
75 nm, and preferably a range from about 25 nm to about 60 nm, are
used for a wide range of fluorophores in some embodiments. In some
embodiments, one or more of the larger thicknesses of the
dielectric layer are chose to enhance a particular fluorophore at
the expense of other fluorophores. Thus, in some embodiments, a
thickness of the dielectric layer is selected to maximize
fluorescent enhancement for a particular fluorophore in the
detection molecule
[0122] For emission enhancement calculations a random distribution
of point dipoles (fluorophores) was assumed both in terms of the
orientation of the transition dipole moments and in distances from
the surface within a 10 nm conformal layer. The total number of
dipoles was 81 in the volume of 100.times.100.times.10 nm.sup.3
with transition moments equally distributed over the X, Y, and Z
direction (27 dipoles per direction). It was found that two-fold
increase of number of dipoles resulted in mirror increase in
calculated emission enhancement of about 5%. The enhancement
factors were calculated relative to the reference system of the
glass slide without any additional dielectric or metal layers. For
the reference sample, only dipoles oriented in the Y direction were
considered.
[0123] FIG. 11A and FIG. 11B are graphs 1100, 1120, respectively,
that illustrate example enhancement of emission light intensity
based on thickness of dielectric layer, according to various
embodiments. The horizontal axis 1102 indicates thickness of the
SiO.sub.2 layer. The vertical axes 1104, 1124, respectively,
indicate the emission enhancement, which is dimensionless. The
measurement of radiation power is within a cone of 18 degrees. FIG.
11A graphs the dipole transition moment parallel to the interface
(Dx, Dy); and FIG. 11B graphs the dipole transition moment
perpendicular to that surface (Dz). On each graph there are five
traces corresponding to five excitation wavelengths. For the two
graphs, respectively, enhancements at 550 nm are shown on traces
1111, 1131; enhancements at 580 nm are shown on traces 1012, 1132;
enhancements at 680 nm are shown on traces 1113, 1133; enhancements
at 720 nm are shown on traces 1114, 1134; and, enhancements at 800
nm are shown on traces 1115, 1135. The dependence of X dipole
emission on silica thickness is similar to the excitation
dependence (FIG. 10A), for example, the enhancements are maximal at
about 25 nm for excitation at 630 nm and for X-oriented emitter at
680 nm. However, the enhancement is quite different for the
Z-oriented emitter; the greatest enhancement is observed for no
silica layer, while some quenching (<1.0) or no enhancement
(.about.1.0) is observed for most of the silica thicknesses.
[0124] The lack of sharp layer-thickness-dependent enhancements is
advantageous for the applications of these Ag film/dielectric
substrates. This is because it allows relaxed conditions for
reproducible fabrication. In addition, it allows use of the same
substrate for a broad range of fluorophores, an important aspect
for multiplexing approaches. These results show that substrates
fabricated using two different vacuum deposition systems, thermal
and magnetron sputtering, resulted in a very similar performance.
It is anticipated that this approach finds immediate applications
in surface-based sensor designs because of easy fabrication and the
availability of a broad range of fluorescent probes for various
biotechnological applications.
[0125] FIG. 12A is a graph 1200 that illustrates example measured
enhancements of fluorescence intensity for an example plasmonic
substrate at multiple optical frequencies, according to various
embodiments. The horizontal axis 1202 indicates individual
fluorophores. The vertical axis 1204 indicate fluorescence
enhancement as a dimensionless factor. Fluorescence enhancements
are depicted for Alexa Fluors and DY dyes in blue-green, including
AF488 as bar 1211, AF532 as bar 1212; in red including AF632 as bar
1213, AF647 as bar 1214, DY649 as bar 1215; in far red including:
AF680 as bar 1216, DY680 as bar 1217; and in near infrared
including: AF750 as bar 1218 and IRD800 as bar 1219. These results
show fluorescence enhancements of more than 200-fold for an
ensemble of dyes in a broad spectral range. Other results (not
plotted) include fluorescence enhancements of about 400-fold to
1000-fold observed for Cy5 single molecules.
[0126] FIG. 12B is a graph 1220 that illustrates example measured
lifetimes of fluorescent emission for an example plasmonic
substrate at multiple optical frequencies, according to various
embodiments. The horizontal axis 1222 indicates 5 individual
fluorophores with and without, respectively, these multiplayer
plasmonic substrates with mirror and dielectric layer. The vertical
axis 1204 indicates lifetime in nanoseconds (ns, 1 ns=10.sup.-9
seconds). Lifetime of every fluorophore is decreased by about
10-fold.
[0127] The excess of the surface area due to Ag nanostructures was
estimated to be less than 25% compared with the planar glass. This
estimate assumed an array of semispherical particles with a
diameter of 40 nm and spacing (side-to-side) of 40 nm and found
that particles will increase the total surface area by about 21%.
Therefore, the observed large fluorescence enhancements are due to
surface enhanced phenomena and not the differences in the surface
concentration of bound dye-streptavidin conjugates. In view of
sensing applications, the increase in the sensing active area is
desirable for improved sensitivity.
[0128] The intensity and lifetime data illustrate that fluorescence
enhancement on multilayer substrates is due to several effects,
including the reflective properties of mirrors with an optimal
dielectric thickness layer, the surface plasmons of the outer layer
of Ag nanostructures, and the increased surface area for protein
binding. It is observed that fluorescence enhancement due to Ag
nanostructures strongly depends on the spectral range. This
suggests that the far-red (AF647) and possibly infrared wavelength
ranges are the most promising for fluorescence enhancement with Ag
nano structures. The proper combination of layers and the annealing
process provides a convenient method for the fabrication of planar
substrates suitable for stable fluorescence enhancements over
200-fold, which can be applied for the design of low-cost detection
instrumentation for proteomics and genomics applications
[0129] Deposition of thin silver films and subsequent thermal
annealing provides a means for the fabrication of silver
nanostructures that significantly amplify the fluorescence. The
enhancement of fluorescence is highly dependent on the fabrication
parameters and the spectral range. An average enhancement from
about 50-fold in blue-green and up to 210-fold in the red range has
been observed. A noted advantage of the fabrication method is its
simplicity. The substrates can be deposited by a vacuum process,
followed by annealing in air. In addition, because of the
simplicity of the method, a large number of substrates can be
produced using simple microscope glass slides that accommodate a
large area for enhanced biosensing and can utilize many available
fluorescent readers and fluorophores. It is expected that high
reproducibility of substrates for enhanced fluorescence are
achieved with the described fabrication process. This is because
the critical thicknesses of the dielectric and Ag outer layers can
be deposited with high accuracy using a vacuum deposition process.
The two vacuum deposition systems used resulted in very similar
values of fluorescence enhancements for two dyes and a broad range
of dielectric thicknesses. Moreover, the optimal fluorescence
enhancement occurs for a relatively broad range of dielectric as
well as Ag effective thicknesses, which further assures the
reproducible performance of the substrates.
[0130] To make use of the mirror's contribution to the enhancement
it is desirable that the optical density of the metal nanoparticles
not be so great as to block transmission of light into the
dielectric layer. Therefore an optical density of less than about
1.0 (10% transmission) is desirable. In other embodiments, optical
density is selected in a range from about 0.2 to 1.5.
2.2 Assays for Cytokines
[0131] FIG. 13 is a block diagram that illustrates example
cytokines of interest, according to various embodiments.
Differentiation of naive CD4+ cells (Thp) into T cell subsets are
defined by their cytokine production profiles. IL-4 drives the
differentiation of Th2 cells, which mainly produce IL-4, IL-5 and
IL-13. IL-12 polarizes Thp cells towards Th1 cells, which mainly
produce IFN.gamma.. The presence of IL-6 and TGF.beta. causes Thp
cells to form into Th17 cells which mainly produce IL-17A, IL-17F,
and IL-22. Th1 cells are considered to be major effectors against
viral infection, intracellular pathogens, and cancers. Recent
studies indicate that detection of double IFN.gamma./IL-2 producing
T cells provides additional clinical information regarding the
prognosis of patients with human immunodeficiency virus (HIV)
compared to enumeration of IFN.gamma. or IL-2 secreting T cells
alone. Furthermore, heterogeneity in IL-5-production by IL-4+Th2
cells was observed in humans and was correlated with different
allergic phenotypes. An association has also been found between the
IL-13+IFN.gamma.+ double producers and enhanced allergic
inflammation.
[0132] In one embodiment, a high density of capture antibody on the
surface of MEF substrate was demonstrated, along with the resulting
intensity enhancement and lifetime decrease of fluorescence of
detection antibody in sandwich cytokine assays. For this
embodiment, results obtained with TNF.alpha. assay and biotinylated
BSA (BSA-Bt) were compared. TNF.alpha. assay comprised of Ab1/TNF
(500 ng/ml)/Ab2-Bt and detection was with dye-streptavidin
conjugates. Phase shifts were measured for probes in solution and
when bound to the surface via captured TNF.alpha.. The results were
compared to biotinylated BSA (BSA-Bt) coated substrates. Blocking
solution (5% BSA) was used to minimize non-specific binding.
Control sample was surface-coated with BSA-Bt where typically a
monolayer of BSA is formed on the surfaces. Probe concentration in
each case was 2 .mu.g/ml, to saturate the biotinylated surface
[0133] FIG. 14 is a graph that illustrates example differences
among lifetimes of fluorescence emissions in various layers in or
above the plasmonic substrate for three dyes, according to an
embodiment. The horizontal axis indicates each of three
fluorophores (AF488, AF636, AF680) in each of three states (free in
solution; in a TNF.alpha. assay; and on the BSA-Bt fixed target
molecule). The vertical axis indicates phase shift at 100 megahertz
(MHz, 1 MHz=10.sup.6 Hertz, Hz, 1 Hz=1 cycle per second) in
degrees. Fluorescence lifetime is related to this phase shift by
.tau.=.omega..sup.-1 tan .phi., where .tau. is lifetime, .omega. is
radial modulation frequency, and .phi. is phase shift. FIG. 14
shows that phase shifts up to almost 45 degrees (significant
lifetime changes) were obtained in the assay configuration. These
large phase changes imply great potential for the design of a
sensitive assay using lifetime detection modality. Larger changes
were observed for BSA-Bt because of the shorter distance between
fluorophore in BSA-Bt/SA-dye compared to Ab1/TNF/Ab2-Bt/SA-dye. The
intensity enhancements in this assay format were slightly smaller
than observed for BSA-Bt of about 20% (not shown). These results
indicate that sandwich assay for TNF.alpha. is not compromised as
compared to BSA-Bt/SA-dye.
[0134] Calibration curves for several cytokines were generated
using commercial reagents for ELISA (all from Pierce Biotechnology)
with secondary biotinylated antibodies (Ab2-Bt) and using
dye-streptavidin conjugates (dye-SA) as detection probes. Briefly,
the procedure involved adsorption of the capture antibody on the
surface by physical adsorption, blocking, adding 3-fold dilutions
of cytokines, incubating with Ab2-Bt, and performing measurements
in the presence or absence of the detection probe. With plasmonic
substrates multiple calibration curves can be generated (while
standard methods allow only single calibration curve at one time).
Because of the high fluorescence amplification of bound probes,
calibration curves can be generated without washing out the
detection probe, which introduces intensity changes and/or lifetime
(phase and modulation) changes. It was found that the most
convenient way to display intensity calibrations is using the ratio
of intensity to the baseline signal. The baseline signal includes
the background from assay matrix (surface and sample reagent),
background from non-specific binding, and the signal from the bulk
detection probe (when not washing). This unified form of
calibration includes information on the signal-to-background noise
(S/N) and allows for direct comparison of various modalities. It is
important to identify what contributes to, e.g. poor performance of
assay, large background signal or poor performance of selected
antibodies.
[0135] FIG. 15A and FIG. 15B are graphs 1500, 1520, respectively,
that illustrate example fluorescence enhancement calibration curves
for two cytokines, according to various embodiments. In graph 1500,
the logarithmic horizontal axis 1502 indicates concentration of
VEGF in nanograms per milliliter (ng/ml, 1 ng=10.sup.-9 grams). The
left side logarithmic vertical axis 1504 indicates intensity ratio,
a dimensionless quantity; and the right side vertical axis 1505
indicates phase shift at 100 MHZ in degrees (which is proportional
to lifetime). Graph 1500 displays three typical intensity
calibration curves when using plasmonic substrates: directly based
on intensity ratio and lifetime (expressed as phase shift) as trace
1513b and 1511; and also intensity after washing out the detection
probe as trace 1513a; and on bare glass as trace 1513c for
reference. Direct calibration curves (intensity and phase shift)
are for no wash of detection probe (AF635-SA). The numbers show
limits of detection (LOD) in ng/ml (baseline plus 2 standard
deviations).
[0136] An advantage of using combined intensity and lifetime
calibrations is large concentration dynamic range of more than 4
decades. This is because the sensitivity of each mode of
calibration is different. A direct method of using phase
measurement shown a trace 1511 displays the best sensitivity. This
is because of favorable weighting of the short lifetime component
from bound probes. For comparison, a standard method using a
commercial glass substrate (need washing out) is included. It is
clear that plasmonic substrates provide significantly better
detection limits and the option of no washing. In a similar way
several cytokine assays were tested (Table 2) and the results are
satisfactory for use in assays. It is evident that the S/N ratio is
excellent for plasmonic substrates allowing detection cytokines in
the range of 10 pg/ml and better are expected for optimized assays
when using dye-labeled antibodies as detection probes.
TABLE-US-00003 TABLE 2 Limit of detection (pg/ml) on MEF substrates
TNFa IFNg IL-8 IL-5 VEGF TNFa Glass 235 380 585 1256 2540 2650 MEF
washed 25 30 15 36 90 210 Direct intensity 75 75 42 235 450 750
Direct phase 6 8 6 9 60 105 Normal serum 1-10 1-10 >10 1-10 ~180
~50 ng/ml
[0137] The performance of MEF substrates is comparable to ELISA and
ELISPOT using cell-free conditions. To carry out biochemical
procedures and imaging, an adhesive silicon gasket was placed which
served as a liquid reservoir for solution exchange and later for
cell wells. The substrate was activated with capture antibodies
immobilized using EDC/NHS procedure followed by blocking solution
(5% BSA). The activated substrate was kept in culture media
RPMI-1640 for 24 hrs in a CO.sub.2 incubator at 37.degree. C. Next,
using dilutions of a mixture of recombinant TNF.alpha. and
IFN.gamma. (0.8 pg/ml-500 ng/ml) and the AF647 labeled detection
antibodies (1 .mu.g/ml each) were added to the wells. After 1 hour
incubation, the wells were imaged with a FLIM instrument. The
intensities were averaged over the entire spots and normalized to
the baseline intensity (area outside the spots).
[0138] In graph 1520, the logarithmic horizontal axis 1522
indicates concentration of IFNg or TNF.alpha. in nanograms per
milliliter. The left side logarithmic vertical axis 1524 indicates
intensity ratio, a dimensionless quantity; and the right side
vertical axis 1525 indicates lifetime in nanoseconds. Graph 1520
illustrates example calibration curves for two cytokines using
Ab2-AF647. The fixed target includes two different capture
antibodies that were spotted as 1 .mu.l drops on different areas of
the substrate. The assay was performed with a mixture of equimolar
concentration of TNF.alpha. and IFN.gamma.. The intensity curves
are IFN.gamma. direct trace 1531, TNF.alpha. direct trace 1532, and
TNF.alpha. with washed probes trace 1533. The lifetime trace 1534
is for TNF.alpha. direct. LODs were determined as baseline plus 2
standard deviations; and were 60 pg/ml for traces 1531 and 1532, 16
pg/ml for trace 1533 and 6 pg/ml for trace 1534.
[0139] The lifetime parameter is more general than the phase shift
because it can be applied to either time-domain or frequency-domain
fluorometry. As shown here, lifetime modality is as highly
sensitive as the phase shift measurements (FIG. 15A). In addition
images were acquired after washing out dye-labeled antibodies, and
a calibration curve generated for TNF.alpha.. FIG. 16A and FIG. 16B
are images 1610 and 1620 that illustrate example spot
quantification of two cytokines with and without washing, according
to various embodiments. Image 1610 illustrates example direct (in
presence of detection probes) active fluorescence spots 1611 for
IFN.gamma. and 1612 for TNF.alpha. at concentration of 0.51 ng/ml.
Clearly there is a significant background signal seen between
active spots that is also present over the active spots but does
not prevent quantification of cytokine concentration at the active
spots. Image 1620 illustrates example washed active fluorescence
spots 1621 for IFN.gamma. and 1622 for TNF.alpha. at concentration
of 0.51 ng/ml after washing out.
[0140] The performance of the assay using various calibration
modalities are characterized with LODs. The obtained sensitivities
which are comparable with ELISA/ELISPOT indicate that the MEFspot
method has great potential for sensing secreted proteins in cell
culture environments.
[0141] Cytokine secretion is usually measured by ELISA in cell
supernatants. This method works well if there is sufficient number
of activated cells to produce cytokine concentration above the
ELISA sensitivity limit, typically 10 pg/ml. The MEFspot quantifies
the local concentration of cytokine(s) and thus, enables the direct
measurement of cytokine secretion from a single cell. To
demonstrate the capability of MEFspot for quantification of
secretion in a cellular environment, human macrophages stimulated
with macrophage colony stimulating factor (MCSF) for 12 hours were
used for detection of secreted TNF.alpha.. In order to test real
sensitivity, different cell numbers per well were used. The
substrate was activated with a capture antibody (EDC/NHS protocol)
and incubated with cells during stimulation. Cell supernatants were
collected for subsequent ELISA measurements.
[0142] FIG. 17A, FIG. 17B and FIG. 17C are images that illustrate
example spot quantification of fluorescence in the presence of
cells for three concentrations of cells, respectively, according to
various embodiments. FIG. 17A, FIG. 17B and FIG. 17C illustrate
example quantification of secreted TNF.alpha. by macrophages
stimulated with MCFS with different cell numbers of 30,000 per
well, 10,000 per well, and 5,000 per well, respectively. Top image
panel, i.e., images 1711, 1721 and 1731, shows cells stained with
Calcein AM for the three different cell numbers. Bottom image
panel, i.e., images 1712, 1722, 1732, shows the normalized
fluorescence from bound Ab2-A647 after washing the unbound
detection antibodies. Calcein AM excitation wavelength is 473 nm
and emission wavelength is 525 nm to 530 nm. The AF647-antibody
pair excitation wavelength is 640 nm and emission wavelength is
>655 nm.
[0143] FIG. 18A is a graph 1800 that illustrates example
calibration curve, according to an embodiment. The logarithmic
horizontal axis 1802 indicates TNF.alpha. concentration in
nanograms per milliliter. The linear vertical axis 1804 indicates
intensity ratio, a dimensionless quantity. Three areas were
considered for quantification of TNF.alpha.: (1) surface between
cells, (2) whole image, and (3) bright spots. The intensities were
normalized to the baseline (non-active area in the wells)
correlated with calibration curve and plotted as symbols on the
calibration curve. The supernatant was used as input to the ELISA
process. Comparisons of readout from various areas and ELISA are
depicted in FIG. 18B.
[0144] FIG. 18B is a graph 1820 that illustrates example advantages
over a prior art approach, according to an embodiment. The
horizontal axis indicates different methods of quantification for
the three cell counts depicted in FIG. 17A through FIG. 17C and
designated (a), (b) and (c) in FIG. 18B. The vertical axis
indicates the TNF.alpha. concentration deduced, in picograms per
milliliter. The results are excellent. There is correlation between
the supernatant and the surface-detected TNF.alpha. for all three
cell counts. The average concentration decreases with a decreased
number of cells. Production of secreted TNF.alpha. per active cell
is larger for the high cell counts (a) that exhibit cell clusters
than low cell counts (c) that exhibit primarily isolated cells, in
agreement with other studies. These data illustrate the potential
of MEFspot for immediate quantification with additional options
compared to only post-experimental ELISA measurements, which may be
not sensitive enough, e.g. for low cell counts (c). This
experimental embodiment also confirmed the ability to elucidate
information that cell clusters play crucial roles in the function
of macrophages. It should be mentioned that MEFspot allows for
imaging (or reimaging) of desired areas including selection of
individual cells with high spatial resolution. In some embodiments,
bright spots due to artifacts are rejected from analysis when
lifetime analysis is included, because such artifacts often produce
longer lifetimes than expected in solution.
[0145] A new and unique feature of the MEFspot is the ability for
real-time monitoring of protein secretion by cells. FIG. 19A, FIG.
19B and FIG. 19C are images 1910, 1920, 1930, respectively, that
illustrate example real-time measurement of time-dependent
secretion of a cytokine from a subset of cells in a population,
according to various embodiments. The intensity gray scale is shown
in bar 1902 for all three images. The highest intensities appear in
image 1930 at the spots indicated by arrows 1932. These images show
the result of monitoring the secretion of TNF.alpha. by macrophages
activated with 30 ng/ml of lipopolysaccharide (LPS) over an eight
hour period. In image 1910, cells are visualized with Calcein AM,
for an image size of 2,000.times.2,000 .mu.m.sup.2. Image 1920
illustrates example intensity of Ab2-AF647 at one hour after
stimulation; and, image 1930 illustrates example intensity at eight
hours after stimulation.
[0146] FIG. 19D is a graph 1940 that illustrates example use of
calibration curve 1951, according to an embodiment. The logarithmic
horizontal axis 1942 indicates TNFa concentration in nanograms per
milliliter. The vertical axis 1944 indicates intensity ratio, which
is dimensionless. As time increases, the intensity ratio
observations signified by points slide up the calibration curve
1951, as signified by the arrow indicating time after start of
stimulation, thus indicating an increase in TNFa concentration with
time. FIG. 19E is a graph 1960 that illustrates example measured
time series of cytokine secretion, according to an embodiment. The
horizontal axis 1962 indicates time of LPS activation in hours;
and, the vertical axis 1964 indicates TNFa concentration in
picograms per milliliter, now on a linear scale. Trace 1971 and
observation points signified by stars are based on mapping the
observed intensity values to concentration values using the
calibration curve 1951 at each time point. Quantification was
performed using average intensities from whole images and
correlated to the calibration curve (in the presence of the
detection probe). The large error bars (one standard deviation) in
the effective TNF.alpha. concentration are due to large differences
between bright spots and dim surface, which reflects variations in
individual (or cell clusters) secretion and average
supernatant.
[0147] A significant increase in cytokine production was observed
over time. One can imagine a low number of active cells and monitor
cytokine production by individual cells with high spatial
resolution. High dye photostability in the presence of plasmonic
substrates allows multiple time imaging without compromising the
brightness of spots.
[0148] A phasor analysis of FLIM data is a very sensitive
"fit-free" method to identify and distinguish different image areas
based on the lifetime. The phasor plot is very convenient and
powerful tool for analyzing FLIM images, and is gaining attention
for cellular studies. The phasor plot is particularly attractive in
MEFspot because of intrinsic changes in the lifetime of the
detection probe upon binding to functionalized plasmonic
substrates. Thus, cells secreting proteins can be visualized based
on the lifetime values and provide an additional imaging tool to
the biologist in interpreting usually complicated intensity images.
The FLIM imager was used with experimental MEFspot assay.
[0149] FIG. 20A, FIG. 20B and FIG. 20C, illustrate example enhanced
analysis of fluorescence data from both intensity and lifetime
measurements, according to an embodiment. FIG. 20A is an image 2010
that illustrates an example FLIM image of intensity of a MEFspot
assay. Highest intensity spots are indicated by arrows 2012.
[0150] Each pixel in the image 2010 is also associated with a
lifetime measurement used to generate a phasor plot. FIG. 20B is a
graph 2020 that illustrates an example phasor plot. The horizontal
axis 2022 indicates a G coordinate of the lifetime information,
which is dimensionless; and the vertical axis 2024 indicates an S
coordinate of the lifetime information, also dimensionless. The S
and G coordinates are the Fourier sine (S) and cosine (G)
transforms related to lifetime components, amplitudes and
modulation frequency. Pixels with single exponential decay
lifetimes are located on the semicircle 2025. For bi-exponential
decays, the locations of the two component phasors are along the
line joining the two lifetime points defined by the fractional
contribution of each component (f.sub.i) in the observed total
intensity. Every pixel of the intensity image is transformed into a
point (pixel) in the phasor plot with coordinates S and G defined
by the lifetime data for the pixel. The pixel locations are on the
line between two single exponential lifetimes depicted as 2.1 ns,
point 2026, and 0.5 ns, point 2027. Three regions on the phasor
plot are selected (2028a, 2028b and 2028c). The long lifetime
pixels (2028a) indicate the area of only free detection probe in
solution. The short lifetime pixels (2028c) show areas that
correspond to high level of TNF-.alpha. secretion. The middle
lifetime pixels (2028b) shows intermediate average lifetime of 1.25
ns.
[0151] FIG. 20C is an image 2030 that illustrates results of the
phasor classification mapped to the spatial arrangement in the
assay. The long lifetime pixels indicated by arrows 2032 locate the
areas of only free detection probe in solution. The short lifetime
pixels are the remaining dark areas on the image 2030. The middle
lifetime pixels are bright areas in image 2030. The phasor plot
allows identification of spots (cells, cell clusters) generated by
secreted TNF.alpha. in a heterogeneous intensity image.
[0152] In some embodiments, simultaneous imaging of cytokine
secretion and cell phenotype is performed. The performance of
plasmonic substrates are determined using HEK293T cells transiently
transfected with Toll-like receptor (TLR) fused with Cerulen
fluorescent protein (Cer). Conventional methods of transient
transfection produce cell population heterogeneous with respect to
expression of transfected protein. A model cell population is
created in which the expression of a function-defining protein in a
single cell is visualized simultaneously with protein secretion by
the same cell. Exogenous expression of TLR in HEK293T cells
promotes a strong cellular response to TLR agonists; e.g., the
cells that express TLR produce cytokines in agonist-dependent
manner. TLR4-Cer and TLR2-Cer are used in various embodiments. TLR4
recognizes bacterial lipopolysaccharides (LPS) and TLR2 senses
bacterial lipoproteins (synthetic lipoproteins are commercially
available).
[0153] FIG. 21 is a graph 2100 that illustrates example correlation
of phenotype with profile of secreted cytokines, according to an
embodiment. The horizontal 2102 axis indicates cell and tag; and,
the vertical axis 2104 indicates intensity in relative units.
TLR4-Cer fusion protein retains intact signaling properties in
HEK293T cells stimulated with LPS. HA-tagged TLR4 and Cer-tagged
TLR4 induce NF-.kappa.B reporter similarly. These results
demonstrate that transfection of HEK293T cells with TLR4-Cer and
stimulated with LPS induces NF-.kappa.B similarly to the
TLR4HA-tagged through the N terminus. In some embodiments, TLR
expression in a single cell is imaged by fluorescence microscopy
and used as the phenotype-defining marker for the population of
cytokine-secreting cells such as TNF.alpha., IFN.gamma. and dual
TNF.alpha./IFN.gamma.. In an example embodiment, a 443 nm laser is
used for Cer; and a 532 nm laser is used for IFN.gamma. and a 640
nm laser is used for TNF.alpha..
[0154] To correlate cell phenotype with cell function at the single
cell level in some embodiments, TLR-Cer-transfected cells are
stimulated by a TLR agonist or treated by the TLR agonist in the
presence of TLR antagonist. This model permits a demonstration of
the feasibility of simultaneous detection of a protein marker
expressed in cell and the cytokine secretion profile of this cell
in a heterogeneous cell population. To further characterize the
capabilities of MEFspot, the level of TLR expression in a single
cell is correlated quantitatively with the rate and kinetics of
cytokine production by the same cell.
[0155] In some embodiments, MEFspot detects dual cytokine secreting
cells immediately ex vivo, which would be highly significant to the
field of cytokine biology. While early in vitro studies
characterized highly polarized helper T-cell subsets capable of
producing their signature cytokines (e.g., Th2 make IL-4, IL-5,
IL-13), it is becoming appreciated that these cells maintain
dynamic plasticity, especially during inflammatory responses in
vivo. For example, increased numbers of Th2 cells that are also
capable of producing TNF.alpha. or IFN.gamma. were correlated with
enhanced allergic lung inflammation. Furthermore, a variety of
tumor cell types including pancreatic cancer and colorectal cancer
can produce IL-4 or IL13. In addition, tumor infiltrating T-cells
can produce IL-4 and other cytokines which can protect tumor cells
from apoptosis while production of IFN.gamma. promotes antitumor
responses.
[0156] An example embodiment is directed to the production of IL-4,
and IL-13 in combination with other cytokines because of the
relationship between double producers and the phenotype of allergic
lung inflammation. Detection of IL-4 is critical because it is
signature Th2 cytokine and is usually expressed less abundantly
than most other cytokines and also is consumed in culture. Thus
absent or low IL-4 expression in an otherwise Th2-dominated
response might reflect the technical difficulties of IL-4 detection
rather than true IL-4.sup.- Th2 cells. Cytokine pairs that are
examined by MEFspot in various embodiments include single IL-4,
IL-5, and IL-13 and dual IL-4/IL-5, IL-4/IL-13, IL-4/IFN-.gamma.,
IL-5/IFN-.gamma., and IL-13/IFN-.gamma.. Cytokines, antibodies, and
dye labeling kits are supplied by several vendors.
[0157] For example, in some embodiments, secretion of dual
cytokines from lymph node and lung cells are imaged as specified
above. For this experiment, an in vivo model is used, which results
in the development of dual producing T-cells. In vivo-primed CD4+ T
cells are transferred into RAG2.sup.-/- and
.gamma..sub.cxRAG2.sup.-/- mice, and are sensitized and challenged
with a model allergen (ovalbumin). Lymph nodes and lungs are
collected 48 hours after the last challenge; and lymph node cells
harvested by mechanical disruption. Lung tissue from each mouse is
digested with 150 U/ml Collagenase and 10 U/ml DNase for 1 hour at
37.degree. C. Lymph node and lung cells are incubated on the
MEFspot plates immediately ex vivo. They are tested either
untreated, as a control, or stimulated on the plate by adding
PMA/ionomycin. Expression of IL-4/IL-5, IL-4/IL-13,
IL-4/IFN-.gamma., IL-5/IFN-.gamma., and IL-13/IFN-.gamma. in these
cells are imaged using the MEFspot method. These experiments are
performed in the presence and absence of dye conjugated anti-CD4
antibody to distinguish CD4+ T-cells in the complex cell
population. This is especially informative in the lung cells
samples where T-cells are in the minority and other cell types such
as mast cells and basophils can produce IL-4 and IL-13.
[0158] This embodiment can validate the MEFspot method to detect
double cytokine producers in complex cell populations derived ex
vivo from an inflammatory environment. It is anticipated that
MEFspot offers a simple biochemical procedure using live cells.
Importantly this approach allows time-dependent monitoring of the
secreted cytokines and a relatively easy way for quantification of
secretion. These new features enhance the approach for cellular
analysis and reveal more information on changes in cytokine
secretion profiles of T-cells as a result of changes in their
environment (RAG2.sup.-/- and .gamma..sub.cxRAG2.sup.-/-). In other
embodiments, this approach is utilized to address cytokine
secretion by cells in other types of inflammatory environments and
in a complex tumor microenvironment. In some embodiments, the
results are compared to the benchmark technologies, intracellular
cytokine staining (ICCS) and ELISA.
3. Computer Hardware Overview
[0159] FIG. 22 is a block diagram that illustrates a computer
system 2200 upon which an embodiment of the invention may be
implemented. Computer system 2200 includes a communication
mechanism such as a bus 2210 for passing information between other
internal and external components of the computer system 2200.
Information is represented as physical signals of a measurable
phenomenon, typically electric voltages, but including, in other
embodiments, such phenomena as magnetic, electromagnetic, pressure,
chemical, molecular atomic and quantum interactions. For example,
north and south magnetic fields, or a zero and non-zero electric
voltage, represent two states (0, 1) of a binary digit (bit). Other
phenomena can represent digits of a higher base. A superposition of
multiple simultaneous quantum states before measurement represents
a quantum bit (qubit). A sequence of one or more digits constitutes
digital data that is used to represent a number or code for a
character. In some embodiments, information called analog data is
represented by a near continuum of measurable values within a
particular range. Computer system 2200, or a portion thereof,
constitutes a means for performing one or more steps of one or more
methods described herein.
[0160] A sequence of binary digits constitutes digital data that is
used to represent a number or code for a character. A bus 2210
includes many parallel conductors of information so that
information is transferred quickly among devices coupled to the bus
2210. One or more processors 2202 for processing information are
coupled with the bus 2210. A processor 2202 performs a set of
operations on information. The set of operations include bringing
information in from the bus 2210 and placing information on the bus
2210. The set of operations also typically include comparing two or
more units of information, shifting positions of units of
information, and combining two or more units of information, such
as by addition or multiplication. A sequence of operations to be
executed by the processor 2202 constitute computer
instructions.
[0161] Computer system 2200 also includes a memory 2204 coupled to
bus 2210. The memory 2204, such as a random access memory (RAM) or
other dynamic storage device, stores information including computer
instructions. Dynamic memory allows information stored therein to
be changed by the computer system 2200. RAM allows a unit of
information stored at a location called a memory address to be
stored and retrieved independently of information at neighboring
addresses. The memory 2204 is also used by the processor 2202 to
store temporary values during execution of computer instructions.
The computer system 2200 also includes a read only memory (ROM)
2206 or other static storage device coupled to the bus 2210 for
storing static information, including instructions, that is not
changed by the computer system 2200. Also coupled to bus 2210 is a
non-volatile (persistent) storage device 2208, such as a magnetic
disk or optical disk, for storing information, including
instructions, that persists even when the computer system 2200 is
turned off or otherwise loses power.
[0162] Information, including instructions, is provided to the bus
2210 for use by the processor from an external input device 2212,
such as a keyboard containing alphanumeric keys operated by a human
user, or a sensor. A sensor detects conditions in its vicinity and
transforms those detections into signals compatible with the
signals used to represent information in computer system 2200.
Other external devices coupled to bus 2210, used primarily for
interacting with humans, include a display device 2214, such as a
cathode ray tube (CRT) or a liquid crystal display (LCD), for
presenting images, and a pointing device 2216, such as a mouse or a
trackball or cursor direction keys, for controlling a position of a
small cursor image presented on the display 2214 and issuing
commands associated with graphical elements presented on the
display 2214.
[0163] In the illustrated embodiment, special purpose hardware,
such as an application specific integrated circuit (IC) 2220, is
coupled to bus 2210. The special purpose hardware is configured to
perform operations not performed by processor 2202 quickly enough
for special purposes. Examples of application specific ICs include
graphics accelerator cards for generating images for display 2214,
cryptographic boards for encrypting and decrypting messages sent
over a network, speech recognition, and interfaces to special
external devices, such as robotic arms and medical scanning
equipment that repeatedly perform some complex sequence of
operations that are more efficiently implemented in hardware.
[0164] Computer system 2200 also includes one or more instances of
a communications interface 2270 coupled to bus 2210. Communication
interface 2270 provides a two-way communication coupling to a
variety of external devices that operate with their own processors,
such as printers, scanners and external disks. In general the
coupling is with a network link 2278 that is connected to a local
network 2280 to which a variety of external devices with their own
processors are connected. For example, communication interface 2270
may be a parallel port or a serial port or a universal serial bus
(USB) port on a personal computer. In some embodiments,
communications interface 2270 is an integrated services digital
network (ISDN) card or a digital subscriber line (DSL) card or a
telephone modem that provides an information communication
connection to a corresponding type of telephone line. In some
embodiments, a communication interface 2270 is a cable modem that
converts signals on bus 2210 into signals for a communication
connection over a coaxial cable or into optical signals for a
communication connection over a fiber optic cable. As another
example, communications interface 2270 may be a local area network
(LAN) card to provide a data communication connection to a
compatible LAN, such as Ethernet. Wireless links may also be
implemented. Carrier waves, such as acoustic waves and
electromagnetic waves, including radio, optical and infrared waves
travel through space without wires or cables. Signals include
man-made variations in amplitude, frequency, phase, polarization or
other physical properties of carrier waves. For wireless links, the
communications interface 2270 sends and receives electrical,
acoustic or electromagnetic signals, including infrared and optical
signals, that carry information streams, such as digital data.
[0165] The term computer-readable medium is used herein to refer to
any medium that participates in providing information to processor
2202, including instructions for execution. Such a medium may take
many forms, including, but not limited to, non-volatile media,
volatile media and transmission media. Non-volatile media include,
for example, optical or magnetic disks, such as storage device
2208. Volatile media include, for example, dynamic memory 2204.
Transmission media include, for example, coaxial cables, copper
wire, fiber optic cables, and waves that travel through space
without wires or cables, such as acoustic waves and electromagnetic
waves, including radio, optical and infrared waves. The term
computer-readable storage medium is used herein to refer to any
medium that participates in providing information to processor
2202, except for transmission media.
[0166] Common forms of computer-readable media include, for
example, a floppy disk, a flexible disk, a hard disk, a magnetic
tape, or any other magnetic medium, a compact disk ROM (CD-ROM), a
digital video disk (DVD) or any other optical medium, punch cards,
paper tape, or any other physical medium with patterns of holes, a
RAM, a programmable ROM (PROM), an erasable PROM (EPROM), a
FLASH-EPROM, or any other memory chip or cartridge, a carrier wave,
or any other medium from which a computer can read. The term
non-transitory computer-readable storage medium is used herein to
refer to any medium that participates in providing information to
processor 2202, except for carrier waves and other signals.
[0167] Logic encoded in one or more tangible media includes one or
both of processor instructions on a computer-readable storage media
and special purpose hardware, such as ASIC 2220.
[0168] Network link 2278 typically provides information
communication through one or more networks to other devices that
use or process the information. For example, network link 2278 may
provide a connection through local network 2280 to a host computer
2282 or to equipment 2284 operated by an Internet Service Provider
(ISP). ISP equipment 2284 in turn provides data communication
services through the public, world-wide packet-switching
communication network of networks now commonly referred to as the
Internet 2290. A computer called a server 2292 connected to the
Internet provides a service in response to information received
over the Internet. For example, server 2292 provides information
representing video data for presentation at display 2214.
[0169] The invention is related to the use of computer system 2200
for implementing the techniques described herein. According to one
embodiment of the invention, those techniques are performed by
computer system 2200 in response to processor 2202 executing one or
more sequences of one or more instructions contained in memory
2204. Such instructions, also called software and program code, may
be read into memory 2204 from another computer-readable medium such
as storage device 2208. Execution of the sequences of instructions
contained in memory 2204 causes processor 2202 to perform the
method steps described herein. In alternative embodiments,
hardware, such as application specific integrated circuit 2220, may
be used in place of or in combination with software to implement
the invention. Thus, embodiments of the invention are not limited
to any specific combination of hardware and software.
[0170] The signals transmitted over network link 2278 and other
networks through communications interface 2270, carry information
to and from computer system 2200. Computer system 2200 can send and
receive information, including program code, through the networks
2280, 2290 among others, through network link 2278 and
communications interface 2270. In an example using the Internet
2290, a server 2292 transmits program code for a particular
application, requested by a message sent from computer 2200,
through Internet 2290, ISP equipment 2284, local network 2280 and
communications interface 2270. The received code may be executed by
processor 2202 as it is received, or may be stored in storage
device 2208 or other non-volatile storage for later execution, or
both. In this manner, computer system 2200 may obtain application
program code in the form of a signal on a carrier wave.
[0171] Various forms of computer readable media may be involved in
carrying one or more sequence of instructions or data or both to
processor 2202 for execution. For example, instructions and data
may initially be carried on a magnetic disk of a remote computer
such as host 2282. The remote computer loads the instructions and
data into its dynamic memory and sends the instructions and data
over a telephone line using a modem. A modem local to the computer
system 2200 receives the instructions and data on a telephone line
and uses an infra-red transmitter to convert the instructions and
data to a signal on an infra-red a carrier wave serving as the
network link 2278. An infrared detector serving as communications
interface 2270 receives the instructions and data carried in the
infrared signal and places information representing the
instructions and data onto bus 2210. Bus 2210 carries the
information to memory 2204 from which processor 2202 retrieves and
executes the instructions using some of the data sent with the
instructions. The instructions and data received in memory 2204 may
optionally be stored on storage device 2208, either before or after
execution by the processor 2202.
[0172] FIG. 23 illustrates a chip set 2300 upon which an embodiment
of the invention may be implemented. Chip set 2300 is programmed to
perform one or more steps of a method described herein and
includes, for instance, the processor and memory components
described with respect to FIG. 22 incorporated in one or more
physical packages (e.g., chips). By way of example, a physical
package includes an arrangement of one or more materials,
components, and/or wires on a structural assembly (e.g., a
baseboard) to provide one or more characteristics such as physical
strength, conservation of size, and/or limitation of electrical
interaction. It is contemplated that in certain embodiments the
chip set can be implemented in a single chip. Chip set 2300, or a
portion thereof, constitutes a means for performing one or more
steps of a method described herein.
[0173] In one embodiment, the chip set 2300 includes a
communication mechanism such as a bus 2301 for passing information
among the components of the chip set 2300. A processor 2303 has
connectivity to the bus 2301 to execute instructions and process
information stored in, for example, a memory 2305. The processor
2303 may include one or more processing cores with each core
configured to perform independently. A multi-core processor enables
multiprocessing within a single physical package. Examples of a
multi-core processor include two, four, eight, or greater numbers
of processing cores. Alternatively or in addition, the processor
2303 may include one or more microprocessors configured in tandem
via the bus 2301 to enable independent execution of instructions,
pipelining, and multithreading. The processor 2303 may also be
accompanied with one or more specialized components to perform
certain processing functions and tasks such as one or more digital
signal processors (DSP) 2307, or one or more application-specific
integrated circuits (ASIC) 2309. A DSP 2307 typically is configured
to process real-world signals (e.g., sound) in real time
independently of the processor 2303. Similarly, an ASIC 2309 can be
configured to performed specialized functions not easily performed
by a general purposed processor. Other specialized components to
aid in performing the inventive functions described herein include
one or more field programmable gate arrays (FPGA) (not shown), one
or more controllers (not shown), or one or more other
special-purpose computer chips.
[0174] The processor 2303 and accompanying components have
connectivity to the memory 2305 via the bus 2301. The memory 2305
includes both dynamic memory (e.g., RAM, magnetic disk, writable
optical disk, etc.) and static memory (e.g., ROM, CD-ROM, etc.) for
storing executable instructions that when executed perform one or
more steps of a method described herein. The memory 2305 also
stores the data associated with or generated by the execution of
one or more steps of the methods described herein.
4. Extensions and Modifications
[0175] In the foregoing specification, the invention has been
described with reference to specific embodiments thereof. It will,
however, be evident that various modifications and changes may be
made thereto without departing from the broader spirit and scope of
the invention. The specification and drawings are, accordingly, to
be regarded in an illustrative rather than a restrictive sense.
Throughout this specification and the claims, unless the context
requires otherwise, the word "comprise" and its variations, such as
"comprises" and "comprising," will be understood to imply the
inclusion of a stated item, element or step or group of items,
elements or steps but not the exclusion of any other item, element
or step or group of items. elements or steps. Furthermore, the
indefinite article "a" or "an" is meant to indicate one or more of
the item, element or step modified by the article.
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