U.S. patent application number 17/701562 was filed with the patent office on 2022-07-07 for nucleic acid hybridization assay.
This patent application is currently assigned to Essenlix Corporation. The applicant listed for this patent is Essenlix Corporation. Invention is credited to Stephen Y. Chou, Wei Ding, Ji Li, Ji Qi, Yufan Zhang.
Application Number | 20220213536 17/701562 |
Document ID | / |
Family ID | |
Filed Date | 2022-07-07 |
United States Patent
Application |
20220213536 |
Kind Code |
A1 |
Chou; Stephen Y. ; et
al. |
July 7, 2022 |
NUCLEIC ACID HYBRIDIZATION ASSAY
Abstract
Provided herein is a method and device for performing a
homogeneous nucleic acid detection assay. The device can contain a
pair of plates where one of the plates comprises (i) surface
amplification surface; and (ii) target-specific nucleic acid probes
that are immobilized on said amplification surface and that
specifically binds to a part of the target nucleic acid; and the
second plate comprises a sample contact area comprising a reagent
storage site that comprises target-specific nucleic acid detection
agents that specifically binds to another part of the target
nucleic acid. In some embodiments, the device can be read without a
washing unbound label from the surface of the device.
Inventors: |
Chou; Stephen Y.;
(Princeton, NJ) ; Ding; Wei; (East Windsor,
NJ) ; Qi; Ji; (Lawrence Township, NJ) ; Zhang;
Yufan; (Princeton, NJ) ; Li; Ji; (Princeton,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Essenlix Corporation |
Monmouth Junction |
NJ |
US |
|
|
Assignee: |
Essenlix Corporation
Monmouth Junction
NJ
|
Appl. No.: |
17/701562 |
Filed: |
March 22, 2022 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16483833 |
Aug 6, 2019 |
|
|
|
PCT/US18/17494 |
Feb 8, 2018 |
|
|
|
17701562 |
|
|
|
|
62456598 |
Feb 8, 2017 |
|
|
|
62457084 |
Feb 9, 2017 |
|
|
|
62456904 |
Feb 9, 2017 |
|
|
|
62457075 |
Feb 9, 2017 |
|
|
|
62459337 |
Feb 15, 2017 |
|
|
|
62459303 |
Feb 15, 2017 |
|
|
|
62459267 |
Feb 15, 2017 |
|
|
|
62460052 |
Feb 16, 2017 |
|
|
|
62460083 |
Feb 16, 2017 |
|
|
|
International
Class: |
C12Q 1/6837 20060101
C12Q001/6837; B01L 3/00 20060101 B01L003/00; B01L 7/00 20060101
B01L007/00; C12Q 1/686 20060101 C12Q001/686; G01N 21/65 20060101
G01N021/65 |
Claims
1. A device for performing a homogeneous nucleic acid detection
assay, comprising a first plate, a second plate, a hinge, spacers,
and an imager, wherein (i) the first plate and the second plate are
movable relative to each other into different configurations,
including an open configuration and a closed configuration, (ii)
the first plate and the second plate each comprises a sample
contact area for contacting a sample that contains or is suspected
of containing a target analyte comprising one or more target
nucleic acids; (iii) the sample contact area of the first plate
comprises a binding site comprising a capture agent or
target-specific nucleic acid probe that is immobilized and that
specifically binds to a part of the target nucleic acid; (iv) one
of the sample contact areas comprises a reagent storage site
comprising a labeled detection agent or target-specific nucleic
acid detection agent that binds to another part of the target
nucleic acid, (v) the spacers have a pillar shape, a predetermined
substantially uniform height, and a predetermined inter-spacer
distance, wherein at least one of the spacers is inside the sample
contact area of one or both of the plates; (vi) one or both of the
plates is flexible, and the thickness of the flexible plate times
the Young's modulus of the flexible plate is in the range 60 to 750
GPa-.mu.m; (vii) the fourth power of the inter-spacer-distance
(ISD) divided by the thickness of the flexible plate (h) and the
Young's modulus (E) of the flexible plate, ISD.sup.4/(hE), is equal
to or less than 5.times.10.sup.6 um.sup.3/GPa; (viii) the plates
are coupled by a hinge that is configured to transition the plates
between an open configuration and a closed configuration; and (ix)
the imager detects a label of the labeled detection agent.
2. The device of claim 1, wherein the sample contact area of the
first plate further comprises a proximity-dependent signal
amplification layer, wherein the capture agent is immobilized on
the surface of the proximity-dependent signal amplification
layer.
3. The device of claim 1 further comprising a thermal cycler.
4. The device of claim 1, wherein, in the closed configuration, the
majority of the target analyte binds to the target-specific nucleic
acid probe in 300 seconds or less.
5. The device of claim 1, wherein, in the closed configuration, the
majority of the target analyte binds to the target-specific nucleic
acid probe in 60 seconds or less.
6. The device of claim 1, wherein the target nucleic acid is a DNA
or RNA comprising one or more selected from the group consisting of
genomic DNA, cfDNA, cDNA ctDNA, mRNA, and miRNA.
7. The device of claim 1, wherein the thickness of the sample in
the closed configuration, the concentration of labels dissolved in
the sample in the closed configuration, and the amplification
factor of the surface amplification layer are configured such that
any of the labels that are bound directly or indirectly to the
probe are visible in the closed configuration without washing away
of the unbound labels.
8. The device of claim 2, wherein the labeled detection agent that
is bound indirectly to the target-specific nucleic acid probe via a
target nucleic acid is visible in less than 60 seconds.
9. The device of claim 2, wherein the storage site is approximately
above the binding site on the first plate in the closed
configuration.
10. The device of claim 1, wherein the signal amplification layer
comprises a disk-coupled dots-on-pillar antenna-array (D2PA).
11. The device of claim 1, wherein the signal amplification layer
comprises a layer of metallic material.
12. The device of claim 2, wherein the proximity-dependent signal
amplification layer comprises a continuous metallic film comprising
a material selected from the group consisting of gold, silver,
copper, aluminum, alloys thereof, and combinations thereof.
13. The device of claim 1, wherein the sample contact area of the
first plate further comprises a metallic material layer that either
locally enhances or acts as a reflector, or both, to enhance an
optical signal.
14. The device of claim 2, wherein the proximity-dependent signal
amplification layer comprises a layer of metallic material and a
dielectric material on top of the metallic material layer, wherein
the target-specific nucleic acid probe is on the dielectric
material.
15. The device of claim 1, wherein the sample contact area of the
first plate further comprises a metallic material layer that is a
uniform metallic layer, nanostructured metallic layer, or a
combination thereof.
16. The device of claim 2, wherein the sample contact area of the
first plate further comprises a site that comprises the
proximity-dependent signal amplification layer but not the
target-specific nucleic acid probe.
17. The device of claim 1, wherein the spacers are fixed on one of
the plates, wherein the spacers regulate the spacing between the
first plate and the second plate in the closed configuration.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
non-provisional application Ser. No. 16/483,833 filed on Aug. 6,
2019, which is a .sctn. 371 national stage application of
International Application PCT/US2018/017494 filed on Feb. 8, 2018,
which claims the benefit of priority to U.S. provisional
application Ser. No. 62/456,598 filed on Feb. 8, 2017 (ESX-032PRV),
62/459,337 filed on Feb. 15, 2017, (ESX-033PRV2) 62/457,084 filed
on Feb. 9, 2017 (ESX-017PRV), 62/459,267 filed on Feb. 15, 2017
(ESX-017PRV2), 62/456,904 filed on Feb. 9, 2017 (ESX-027PRV),
62/459,303 filed on Feb. 15, 2017 (ESX-027PRV2), 62/457,075 filed
on Feb. 9, 2017 (ESX-035PRV), 62/460,052 filed on Feb. 16, 2017
(ESX-035PRV2) and 62/460,083 filed on Feb. 16, 2017 (ESX-035PRV3),
the contents of which are relied upon and incorporated herein by
reference in their entirety. The entire disclosure of any
publication or patent document mentioned herein is entirely
incorporated by reference.
FIELD
[0002] Among other things, the present invention is related to
devices and methods of performing biological and chemical assays,
such as but not limited to assays.
BACKGROUND
[0003] Traditional nucleic acid hybridization assay is complex,
time-consuming, laborious and requires lab setups and significant
amount of sample. For example, Southern Blot usually takes a few
hours to complete. In addition, traditional nucleic acid
hybridization assays require a relatively large volume of sample
(typically >100 uL) that is not applicable in many situations in
which samples are limited or scarce. Therefore, it is desirable to
develop a fast, accurate, portable, and/or inexpensive nucleic acid
hybridization assay that requires as little sample as possible. In
addition, it is desirable that the assay can be conducted by a
non-professional. The current invention satisfies these needs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0004] The skilled artisan will understand that the drawings,
described below, are for illustration purposes only. The drawings
are not intended to limit the scope of the present teachings in any
way. The drawings not are not entirely in scale. In the figures
that present experimental data points, the lines that connect the
data points are for guiding a viewing of the data only and have no
other means.
[0005] FIG. 1A-FIG. 1C shows an embodiment of a QMAX (Q:
quantification; M: magnifying; A: adding reagents; X: acceleration;
also known as compressed regulated open flow (CROF)) device, which
comprises a first plate and a second plate. FIG. 1A shows the
perspective view of the plates in an open configuration when the
plates are separated apart; FIG. 1B shows the perspective view and
a sectional view of depositing a sample on the first plate at the
open configuration; FIG. 1C shows the perspective view and a
sectional view of the QMAX device in a closed configuration.
[0006] FIG. 2A-FIG. 2H is an illustration of an exemplary nucleic
acid hybridization assay according to some embodiments of the
present invention.
[0007] FIG. 3(A)-FIG. 3(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0008] FIG. 4 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 3(A)-FIG. 3(C).
[0009] FIG. 5(A)-FIG. 5(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0010] FIG. 6 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 5(A)-FIG. 5(C).
[0011] FIG. 7(A)-FIG. 7(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0012] FIG. 8 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 7(A)-FIG. 7(C).
[0013] FIG. 9(A)-FIG. 9(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0014] FIG. 10 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 9(A)-FIG. 9(C).
[0015] FIG. 11(A)-FIG. 11(D) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0016] FIG. 12 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 11(A)-FIG. 11(D).
[0017] FIG. 13(A)-FIG. 13(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0018] FIG. 14 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 13(A)-FIG. 13(C).
[0019] FIG. 15(A)-FIG. 15(D) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0020] FIG. 16 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 15(A)-FIG. 15(D).
[0021] FIG. 17(A)-FIG. 17(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0022] FIG. 18 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 17(A)-FIG. 17(C).
[0023] FIG. 19(A)-FIG. 19(D) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0024] FIG. 20 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 19(A)-FIG. 19(D).
[0025] FIG. 21(A)-FIG. 21(D) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0026] FIG. 22 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 21(A)-FIG. 21(D).
[0027] FIG. 23(A)-FIG. 23(D) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0028] FIG. 24 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 23(A)-FIG. 23(D).
[0029] FIG. 25(A)-FIG. 25(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0030] FIG. 26 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 25(A)-FIG. 25(C).
[0031] FIG. 27(A)-FIG. 27(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0032] FIG. 28 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 27(A)-FIG. 27(C).
[0033] FIG. 29(A)-FIG. 29(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0034] FIG. 30 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 29(A)-FIG. 29(C).
[0035] FIG. 31(A)-FIG. 31(C) shows an exemplary assay scheme of a
nucleic acid hybridization assay of the present invention.
[0036] FIG. 32 shows an exemplary design of miR21 nucleic acid
sequences and mismatch sequences.
[0037] FIG. 33 shows the signal intensities and changes based on
the design of FIG. 32 and assay of FIG. 31(A)-FIG. 31(C).
[0038] FIG. 34 shows the results of a nucleic acid hybridization
assay based on the scheme as shown in FIG. 31(A)-FIG. 31(C).
[0039] FIG. 35a-FIG. 35c shows how the device can be implemented
using spacers.
[0040] FIG. 36a-FIG. 36b is schematic drawings for exemplary
embodiments of wells on first plate of QMAX. FIG. 36a shows a top
view of wells on first plate with (i) round shape with square
lattice (ii) rectangle shape with square lattice (iii) triangle
shape with hexagonal lattice (iv) round shape with aperiodicity.
FIG. 36b shows a top view of well array on first plate with (i) no
metal coating (ii) metal coating on bottom of the well (iii) metal
coating on side wall of the well (iv) metal coating on both bottom
and side wall of the well.
[0041] FIG. 37 is a flow chart showing an implementation of the
method.
[0042] FIG. 38 shows an example of first plate preparation step for
nucleic acid sequencing.
[0043] FIG. 39 is a schematic drawing for an exemplary embodiment
of a QMAX device in a closed configuration for capturing target
nucleic acid.
[0044] FIG. 40 shows representative a time course study for
capturing target DNA with (a) QMAX using 1 pM concentration 1 uL
target nucleic acid sample.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0045] The following detailed description illustrates some
embodiments of the invention by way of example and not by way of
limitation. The section headings and any subtitles used herein are
for organizational purposes only and are not to be construed as
limiting the subject matter described in any way. The contents
under a section heading and/or subtitle are not limited to the
section heading and/or subtitle, but apply to the entire
description of the present invention.
[0046] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present claims are not entitled to antedate such publication by
virtue of prior invention. Further, the dates of publication
provided can be different from the actual publication dates which
can need to be independently confirmed.
QMAX Device and Assay
[0047] In biological and chemical assaying (i.e. testing), a device
and/or a method that simplifies assaying operation or accelerates
assaying speed is often of great value.
[0048] In the QMAX (Q: quantification; M: magnifying; A: adding
reagents; X: acceleration; also known as compressed regulated open
flow (CROF)) assay platform, a QMAX card uses two plates to
manipulate the shape of a sample into a thin layer (e.g. by
compressing) (as illustrated in FIG. 1). In certain embodiments,
the plate manipulation needs to change the relative position
(termed: plate configuration) of the two plates several times by
human hands or other external forces. There is a need to design the
QMAX card to make the hand operation easy and fast.
[0049] In QMAX assays, one of the plate configurations is an open
configuration, wherein the two plates are completely or partially
separated (the spacing between the plates is not controlled by
spacers) and a sample can be deposited. Another configuration is a
closed configuration, wherein at least part of the sample deposited
in the open configuration is compressed by the two plates into a
layer of highly uniform thickness, the uniform thickness of the
layer is confined by the inner surfaces of the plates and is
regulated by the plates and the spacers.
[0050] In a QMAX assay operation, an operator needs to first make
the two plates to be in an open configuration ready for sample
deposition, then deposit a sample on one or both of the plates, and
finally close the plates into a close position. In certain
embodiments, the two plates of a QMAX card are initially on top of
each other and need to be separated to get into an open
configuration for sample deposition. When one of the plate is a
thin plastic film (175 um thick PMMA), such separation can be
difficult to perform by hand. The present invention intends to
provide the devices and methods that make the operation of certain
assays, such as the QMAX card assay, easy and fast.
[0051] One aspect of the present invention is to have a hinge that
connect two or more plates together, so that the plates can open
and close in a similar fashion as a book.
[0052] Another aspect of the present invention is to configure the
material of the hinge, such that the hinge can self-maintain the
angle between the plates after adjustment.
[0053] Another aspect of the present invention is to configure the
material of the hinge, which maintain the QMAX card in the closed
configuration, such that the entire QMAX card can be slide in and
slide out a card slot without causing accidental separation of the
two plates.
[0054] Another aspect of the present invention is to provide
opening mechanisms such as but not limited to notches on plate
edges or strips attached to the plates, making is easier for a user
to manipulate the positioning of the plates, such as but not
limited to separating the plates of by hand.
[0055] Another aspect of the present invention is to provide a
hinge that can control the rotation of more than two plates.
[0056] The term "compressed open flow (COF)" refers to a method
that changes the shape of a flowable sample deposited on a plate by
(i) placing other plate on top of at least a part of the sample and
(ii) then compressing the sample between the two plates by pushing
the two plates towards each other; wherein the compression reduces
a thickness of at least a part of the sample and makes the sample
flow into open spaces between the plates. The term "compressed
regulated open flow" or "CROF" (or "self-calibrated compressed open
flow" or "SCOF" or "SCCOF") (also known as QMAX) refers to a
particular type of COF, wherein the final thickness of a part or
entire sample after the compression is "regulated" by spacers,
wherein the spacers are placed between the two plates. Here the
CROF device is used interchangeably with the QMAX device.
[0057] The term "spacers" or "stoppers" refers to, unless stated
otherwise, the mechanical objects that set, when being placed
between two plates, a limit on the minimum spacing between the two
plates that can be reached when compressing the two plates
together. Namely, in the compressing, the spacers will stop the
relative movement of the two plates to prevent the plate spacing
becoming less than a preset (i.e. predetermined) value.
[0058] The term "a spacer has a predetermined height" and "spacers
have a predetermined inter-spacer distance" means, respectively,
that the value of the spacer height and the inter spacer distance
is known prior to a QMAX process. It is not predetermined, if the
value of the spacer height and the inter-spacer distance is not
known prior to a QMAX process. For example, in the case that beads
are sprayed on a plate as spacers, where beads are landed at random
locations of the plate, the inter-spacer distance is not
predetermined. Another example of not predetermined inter spacer
distance is that the spacers moves during a QMAX processes.
[0059] The term "a spacer is fixed on its respective plate" in a
QMAX process means that the spacer is attached to a location of a
plate and the attachment to that location is maintained during a
QMAX (i.e. the location of the spacer on respective plate does not
change) process. An example of "a spacer is fixed with its
respective plate" is that a spacer is monolithically made of one
piece of material of the plate, and the location of the spacer
relative to the plate surface does not change during the QMAX
process. An example of "a spacer is not fixed with its respective
plate" is that a spacer is glued to a plate by an adhesive, but
during a use of the plate, during the QMAX process, the adhesive
cannot hold the spacer at its original location on the plate
surface and the spacer moves away from its original location on the
plate surface.
[0060] The term "open configuration" of the two plates in a QMAX
process means a configuration in which the two plates are either
partially or completely separated apart and the spacing between the
plates is not regulated by the spacers
[0061] The term "closed configuration" of the two plates in a QMAX
process means a configuration in which the plates are facing each
other, the spacers and a relevant volume of the sample are between
the plates, the relevant spacing between the plates, and thus the
thickness of the relevant volume of the sample, is regulated by the
plates and the spacers, wherein the relevant volume is at least a
portion of an entire volume of the sample.
[0062] The term "a sample thickness is regulated by the plate and
the spacers" in a QMAX process means that for a give condition of
the plates, the sample, the spacer, and the plate compressing
method, the thickness of at least a port of the sample at the
closed configuration of the plates can be predetermined from the
properties of the spacers and the plate.
[0063] The term "inner surface" or "sample surface" of a plate in a
QMAX device refers to the surface of the plate that touches the
sample, while the other surface (that does not touch the sample) of
the plate is termed "outer surface".
[0064] The term "height" or "thickness" of an object in a QMAX
process refers to, unless specifically stated, the dimension of the
object that is in the direction normal to a surface of the plate.
For example, spacer height is the dimension of the spacer in the
direction normal to a surface of the plate, and the spacer height
and the spacer thickness means the same thing.
[0065] The term "area" of an object in a QMAX process refers to,
unless specifically stated, the area of the object that is parallel
to a surface of the plate. For example, spacer area is the area of
the spacer that is parallel to a surface of the plate.
[0066] The term of QMAX device refers the device that perform a
QMAX (e.g. CROF) process on a sample, and have or not have a hinge
that connect the two plates.
[0067] The term "QMAX device with a hinge and "QMAX card" are
interchangeable.
[0068] The term "angle self-maintain", "angle self-maintaining", or
"rotation angle self-maintaining" refers to the property of the
hinge, which substantially maintains an angle between the two
plates, after an external force that moves the plates from an
initial angle into the angle is removed from the plates.
[0069] The term "proximity-dependent signal amplification layer",
"proximity-dependent signal amplification layer", or "surface
signal amplification layer/surface" refers to a signal
amplification layer that amplifies a signal from an analyte or a
labeled analyte (e.g., a light-emitting label) in a
proximity-dependent manner. In use of such a layer, the signal from
an analyte or a labeled analyte increases the closer the molecule
is to the surface of the signal amplification layer. As would be
apparent, the magnitude of the signal produced by a first labeled
molecule that is proximal to such a layer will be higher than the
signal produced by a second labeled molecule that is distal to the
layer. For example, the signal of a labeled molecule that is within
100 nm of a proximity-dependent signal amplification layer is
greater than the signal of a labeled molecule that is 1 um or more
away from the proximity-dependent amplification layer.
[0070] Signals can be detected using both "lump-sum" and
"pixel-counting" methods. Lump sum methods are those in which the
total signal produced by multiple binding events is determined.
Pixel-counting methods are those that identify individual binding
events and count them digitally.
[0071] In certain embodiments, the QMAX device is configured to
have a detection sensitivity of 0.1 nM or less, such as 10 pM or
less, or 1 pM or less, or 100 fM or less, such as 10 fM or less,
including 1 fM or less, or 0.5 fM or less, or 100 aM or less, or 50
aM or less, or 20 aM or less. In certain embodiments, the QMAX
device is configured to have a detection sensitivity in the range
of 10 aM to 0.1 nM, such as 20 aM to 10 pM, 50 aM to 1 pM,
including 100 aM to 100 fM. In some instances, the QMAX device is
configured to be able to detect analytes at a concentration of 1
ng/mL or less, such as 100 pg/mL or less, including 10 pg/mL or
less, 1 pg/mL or less, 100 fg/mL or less, 10 fg/mL or less, or 5
fg/mL or less. In some instances, the QMAX device is configured to
be able to detect analytes at a concentration in the range of 1
fg/mL to 1 ng/mL, such as 5 fg/mL to 100 pg/mL, including 10 fg/mL
to 10 pg/mL. In certain embodiments, the QMAX device is configured
to have a dynamic range of 5 orders of magnitude or more, such as 6
orders of magnitude or more, including 7 orders of magnitude or
more.
Examples of Wash-free Homogenous QMAX Devices
[0072] In these embodiments, near the top surface of substrate,
there is an amplification zone, where only the label binding or
very close to the substrates got enhanced.
[0073] The plates are moveable relative to each other into
different configuration. One of the configurations is an open
configuration, in which the two plates are partially or entirely
separated apart and the spacing between the plates are not
regulated by the spacers. In some embodiments, the inner surface of
a respective plate comprises a sample contact area, which occupies
a part of the entirety of the inner surface. In certain
embodiments, the spacers are positioned within the sample contact
area. In some embodiments, the spacers are not fixed to any one of
the plates, but are mixed in the sample.
[0074] The sample is any liquid that needs testing. In some
embodiments, the sample is a body fluid that is with or without
processing or dilution. For example, the body fluid can be whole
blood, blood plasma, serum, urine, saliva, sweat, or breath
condensate. In some embodiments, the sample is blood. In certain
embodiments, the sample comprises plasma. In certain embodiments,
the sample comprises whole blood. In certain embodiments, the
sample is a blood or plasma that has been diluted with buffer for
0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90,
100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 5,000, 10,000,
50,000, 100,000, 500,000, or 1,000,000 times or in a range between
any of the two values. In some embodiments, the sample comprises a
target nucleic acid of any sequence, e.g., cfDNA, ctDNA, cfRNA,
mirna, etc.
[0075] Without any intention to limit the use of the present method
and device, in some embodiments, the method may be employed to
identify a microbial pathogen from a clinical sample. In these
embodiments, the target sequences may be from multiple different
pathogens (e.g., at least 10 or at least 100 different pathogens),
without knowing which pathogen is responsible for an infection,
Microbes that might be identified using the present methods,
compositions and kits include but are not limited to: a plurality
of species of Gram (+) bacteria, plurality of species of Gram (-)
bacteria, a plurality of species of bacteria in the family
Enterobacteriaceae, a plurality of species of bacteria in the genus
Enterococcus, a plurality of species of bacteria in the genus
Staphylococcus, and a plurality of species of bacteria in the genus
Campylobacter, Escherichia coli (E. coli), E. coli of various
strains such as, K12-MG1655, CFT073, O157:H7 EDL933, O157:H7
VT2-Sakai, etc., Streptococcus pneumoniae, Pseudomonas aeruginosa,
Staphylococcus aureus, coagulase-negative staphylococci, a
plurality of candida species including C. albicans, C. tropicalis,
C. dubliniensis, C. viswanathii, C. parapsilosis, Klebsiella
pneumoniae, a plurality of Mycobacterium species such as M.
tuberculosis, M. bovis, M. bovis BCG, M. scrofulaceum, M. kansasii,
M. chelonae, M. gordonae, M. ulcerans, M. genavense, M. xenoi, M.
simiae, M. fortuitum, M. malmoense, M. celatum, M. haemophilum and
M. africanum, Listeria species, Chlamydia species, Mycoplasma
species, Salmonella species, Brucella species, Yersinia species,
etc. Thus, the subject method enables identification of microbes to
the level of the genus, species, sub-species, strain or variant of
the microbe.
[0076] In some embodiments, examples of target nucleic acid
sequences in sample may be from Bacillus anthracis (LF), Giardia
lamblia, Legionella, Total Coliforms (including fecal coliform and
E. Coli), Viruses (enteric) stapylococci (e.g., Staphylococcus
epidermidis and Staphylococcus aureus (enterotoxin A, B, C, G, I,
cells, TSST-1), Enterrococcus faecalis, Pseudomonas aeruginosa,
Escherichia coli (Shiga-like toxin, F4, F5, H, K, O, bacteriophage
K1, K5, K13), other gram-positive bacteria, and gram-negative
bacilli. Clostridium difficile, Bacteroidetes, Cryptosporidium
parvum (GP900, p68 or cryptopain, oocyst), Candida albicans,
Bacillus anthracis, Bacillus stearothermophilus, Bacillus cereus,
Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus,
Bacillus badius, Bacillus globigii, Salmonella typhimurium,
Escherichia coli O157:H7, Norovirus, Listeria monocytogenes,
Leptospira interrogans, Leptospira biflexa, Campylobacter jejuni,
Campylobacter coli, Clostridium perfringens, Aspergillus flavus,
Aspergillus parasiticus, Ebola virus, Histoplasma capsulatum,
Blastomyces dermatitidis, Gram-positive bacteria, Gram-negative
bacteria (such as Pseudomonas aeruginosa, Klebsiella pneumoniae,
Salmonella enteriditis, Enterobacter aerogenes,
Enterobacterhermanii, Yersinia enterocolitica and Shigella sonnei),
Polio virus, Influenza type A virus, Disease specific prion
(PrP-d), Hepatitis A virus, Toxoplasma gondii, Vibrio cholera,
Vibrio parahaemolyticus, Vibrio vulnificus, Enterococcus faecalis,
Enterococcus faecium.
[0077] Other pathogens that can be detected in a diagnostic sample
using the devices, systems and methods in the present invention
include, but are not limited to: Varicella zoster; Staphylococcus
epidermidis, Escherichia coli, methicillin-resistant Staphylococcus
aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis,
Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus
capitis, Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus
influenzae, Staphylococcus simulans, Streptococcus pneumoniae and
Candida albicans; gonorrhea (Neisseria gorrhoeae), syphilis
(Treponema pallidum), clamydia (Clamyda tracomitis), nongonococcal
urethritis (Ureaplasm urealyticum), chancroid (Haemophilus
ducreyi), trichomoniasis (Trichomonas vaginalis); Pseudomonas
aeruginosa, methicillin-resistant Staphlococccus aureus (MSRA),
Klebsiella pneumoniae, Haemophilis influenzae, Staphylococcus
aureus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae,
Escherichia coli, Enterococcus faecalis, Serratia marcescens,
Haemophilis parahaemolyticus, Enterococcus cloacae, Candida
albicans, Moraxiella catarrhalis, Streptococcus pneumoniae,
Citrobacter freundii, Enterococcus faecium, Klebsella oxytoca,
Pseudomonas fluorscens, Neiseria meningitidis, Streptococcus
pyogenes, Pneumocystis Klebsella pneumoniae Legionella pneumophila,
Mycoplasma pneumoniae, and Mycobacterium tuberculosis, etc.,
[0078] In particular embodiments, the sample may be obtained from a
biological sample such as cells, tissues, bodily fluids, and stool.
Bodily fluids of interest include but are not limited to, amniotic
fluid, aqueous humour, vitreous humour, blood (e.g., whole blood,
fractionated blood, plasma, serum, etc.), breast milk,
cerebrospinal fluid (CSF), cerumen (earwax), chyle, chime,
endolymph, perilymph, feces, gastric acid, gastric juice, lymph,
mucus (including nasal drainage and phlegm), pericardial fluid,
peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin
oil), semen, sputum, sweat, synovial fluid, tears, vomit, urine and
exhaled condensate. In particular embodiments, a sample may be
obtained from a subject, e.g., a human, and it may be processed
prior to use in the subject assay. For example, prior to analysis,
the protein/nucleic acid may be extracted from a tissue sample
prior to use, methods for which are known. In particular
embodiments, the sample may be a clinical sample, e.g., a sample
collected from a patient.
[0079] The label is a light-emitting label or an optical detectable
label, directly or indirectly, either prior to or after it is bound
to said capture agent. The label is label with signal of Raman
scattering, chromaticity, luminescence, fluorescence,
electroluminescence, chemiluminescence, and/or
electrochemiluminescence. As used herein, the term "light-emitting
label" refers to a label that can emit light when under an external
excitation. This can be luminescence. Fluorescent labels (which
include dye molecules or quantum dots), and luminescent labels
(e.g., electro- or chemi-luminescent labels) are types of
light-emitting label. The external excitation is light (photons)
for fluorescence, electrical current for electroluminescence and
chemical reaction for chemi-luminscence. An external excitation can
be a combination of the above. The phrase "labeled analyte" refers
to an analyte that is detectably labeled with a light emitting
label such that the analyte can be detected by assessing the
presence of the label. A labeled analyte may be labeled directly
(i.e., the analyte itself may be directly conjugated to a label,
e.g., via a strong bond, e.g., a covalent or non-covalent bond), or
a labeled analyte may be labeled indirectly (i.e., the analyte is
bound by a secondary capture agent that is directly labeled).
[0080] The amplification layer amplifies a signal from the target
analyte or a label of the target analyte when the target analyte or
label is 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80
nm, 90 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 1 um, 2 um, 5
um, 10 um from the amplification layer, or a range between any two
of the values; and a preferred range of 1 nm to 50 nm, 50 nm to 100
nm, 100 nm to 200 nm, 200 nm to 500 nm.
[0081] The term "amplify" refers to an increase in the magnitude of
a signal, e.g., at least a 10-fold increase, at least a 100-fold
increase at least a 1,000-fold increase, at least a 10,000-fold
increase, or at least a 100,000-fold increase in a signal.
[0082] In some embodiments, the proximity-dependent signal
amplification layer includes, but not limited to, the
proximity-dependent signal amplification layers described in U.S.
Provisional Patent Application No. 61/347,178, which was filed on
May 21, 2010, U.S. Provisional Patent Application No. 61/622,226,
which was filed on Apr. 10, 2012, U.S. Provisional Patent
Application No. 61/708,314, which was filed on Oct. 1, 2012, U.S.
Provisional Patent Application No. 61/800,915, which was filed on
Mar. 15, 2013, U.S. Provisional Patent Application No. 61/801,933,
which was filed on Mar. 15, 2013, U.S. Provisional Patent
Application No. 61/801,096, which was filed on Mar. 15, 2013, U.S.
Provisional Patent Application No. 61/801,424, which was filed on
Mar. 15, 2013, U.S. Provisional Patent Application No. 61/794,317,
which was filed on Mar. 15, 2013, U.S. Provisional Patent
Application No. 62/090,299, which was filed on Dec. 10, 2014, U.S.
Provisional Patent Application No. 62/066,777, which was filed on
Oct. 21, 2014, U.S. Provisional Patent Application No. 62/234,538,
which was filed on Sep. 29, 2015, U.S. Utility patent application
Ser. No. 13/699,270, which was filed on Jun. 13, 2013, U.S. Utility
patent application Ser. No. 13/838,600, which was filed on Mar. 15,
2013, U.S. Utility patent application Ser. No. 14/459,239, which
was filed on Aug. 13, 2014, U.S. Utility patent application Ser.
No. 14/459,251, which was filed on Aug. 13, 2014, U.S. Utility
patent application Ser. No. 14/852,412, which was filed on Mar. 16,
2014, U.S. Utility patent application Ser. No. 14/871,678, which
was filed on Sep. 30, 2015, U.S. Utility patent application Ser.
No. 14/431,266, which was filed on Oct. 5, 2015, U.S. Utility
patent application Ser. No. 14/668,750, which was filed on Mar. 25,
2015, U.S. Utility patent application Ser. No. 14/775,634, which
was filed on Sep. 11, 2015, U.S. Utility patent application Ser.
No. 14/775,638, which was filed on Sep. 11, 2015, U.S. Utility
patent application Ser. No. 14/852,417, which was filed on Sep. 11,
2015, U.S. Utility patent application Ser. No. 14/964,394, which
was filed on Dec. 9, 2015, PCT Application (designating U.S.) No.
PCT/US2011/037455, which was filed on May 20, 2011, PCT Application
(designating U.S.) No. PCT/US2013/032347, which was filed on Mar.
15, 2013, PCT Application (designating U.S.) No. PCT/US2013/062923,
which was filed on Oct. 1, 2013, PCT Application (designating U.S.)
No. PCT/US2014/030108, which was filed on Mar. 16, 2014, PCT
Application (designating U.S.) No. PCT/US2014/029675, which was
filed on Mar. 14, 2014, PCT Application (designating U.S.) No.
PCT/US2014/028417, which was filed on Mar. 14, 2014, PCT
Application (designating U.S.) No. PCT/US2014/029979, which was
filed on Mar. 15, 2014, PCT Application (designating U.S.) No.
PCT/US2015/056518, which was filed on Oct. 20, 2015, PCT
Application (designating U.S.) No. PCT/US2016/054025, which was
filed on Sep. 27, 2016, the complete disclosures of which are
hereby incorporated by reference for all purposes.
[0083] The signal amplification layer may comprise a continuous
metallic film that is made of a material selected from the group
consisting of gold, silver, copper, aluminum, alloys thereof, and
combinations thereof. The signal amplification layer comprises
high-amplification regions and low-amplification regions, wherein
the high-amplification regions amplify signals at said surface more
than the low-amplification regions, wherein the low-amplification
regions of the layer have been selectively masked, wherein the
signal amplification layer comprises (i) two or more protrusions,
(ii) two or more metal metallic structures, and (iii) two or more
gaps between the metallic structures; thereby increasing the
probability that a target analyte will bind to a high-amplification
region and be detected.
[0084] The signal amplification layer may comprise: [0085] (i) a
substantially continuous metallic backplane on the substrate;
[0086] (ii) one or a plurality of dielectric or semiconductor
pillars extending from the metallic backplane or from the substrate
through holes in the backplane; and [0087] (iii) a metallic disk on
top of the pillar, wherein at least one portion of the edge of the
disk is separated from the metallic backplane by a gap;
[0088] wherein the gap(s) and portion of the metal edges are a part
of the high signal amplification area, wherein the metallic disk
has a shape selected from the group of shapes consisting of round,
polygonal, pyramidal, elliptical, elongated bar shaped, or any
combination thereof. The metallic disc is separated from the
metallic film by a distance in the range of 0.5 to 30 nm, and the
average lateral dimension of the discs is in the range of 20 nm to
250 nm; wherein the signal amplification layer comprises one or
more metallic discs has a shape selected from the group of shapes
consisting of round, polygonal, pyramidal, elliptical, elongated
bar shaped, or any combination thereof, wherein the average lateral
dimension of the discs is in the range 20 nm to 250 nm, and the gap
between adjacent discs in the range of 0.5 to 30 nm.
[0089] wherein the metallic structures are made of a material that
is selected from the group consisting of gold, silver, copper,
aluminum, alloys thereof, and combinations thereof.
[0090] wherein the pillars are periodic or aperiodic, or the
metallic structures have a random shape.
[0091] wherein the signal that is amplified is Raman scattering,
chromaticity, luminescence, fluorescence, electroluminescence,
chemiluminescence, and/or electrochemiluminescence.
[0092] QMAX device's first plate may further comprise a molecular
linking layer that links said capture agents with said signal
amplification layer, wherein said molecular adhesion layer is a
self-assembled monolayer (SAM), wherein each molecule of the SAM
comprises three parts: (i) a head group that has specific affinity
to the signal amplification layer, (ii) a terminal group that
specific affinity to the capture agent, and (iii) a linker that
links the head group and terminal group, wherein the length of the
linker determines the average spacing between the metal signal
amplification layer and an attached capture agent can affects light
amplification of the device.
[0093] QMAX device's second plate sample contact area may comprise
a storage site containing detection agents that upon contacting the
sample, dissolves into the sample and diffuses in the sample,
wherein each capture agent, target analyte and corresponding
detection agent is capable of forming a capture agent-target
analyte-detection agent sandwich in a binding site of the first
plate.
[0094] The device of any prior paragraph, wherein the second plate
sample contact area comprises a storage site containing detection
agents that upon contacting the sample, dissolves into the sample
and diffuses in the sample, wherein the detection agent binds to
the capture agent and competitively inhibits the binding between
the capture agent and the target analyte.
[0095] In some embodiments, the enhancement mechanism of
fluorescence label is known as Plasmonic enhancement. The enhanced
fluorescence intensity due to the proximity of metal nanostructures
makes it possible to detect much lower concentrations of biomarkers
tagged with fluorescence molecule either in sensing format or for
tissue imaging. Metal enhanced fluorescence (MEF) arises from an
increased excitation rate due to an enhanced local field
experienced by the fluorophore, and the electromagnetic coupling of
the fluorophore with the near-by metal nanoparticle. Therefore,
metal nanostructures are able to produce desirable effects such as
increased fluorescence quantum yield, decreased lifetime and better
fluorophore photostability. During the past decade a number of
existing and novel nanoparticles and structures have appeared in
the literature designed to improve both the fluorescence intensity
and photo stability of fluorophores through MEF. Metal
nanostructures have long been researched due to their ability to
manipulate incident light. Localised surface plasmons (LSP) are
charge density oscillations confined to metallic nanostructures and
nanoparticles. If a particle is considered then an external field
is able to displace the free electrons in the metal nanoparticle
with respect to the fixed ionic core. This displacement sets up a
restoring force leading to coherent oscillations of the charge
density. This is termed the Localised Surface Plasmon Resonance
(LSPR). LSPR is responsible for the electromagnetic-field
enhancement that is thought to lead to surface enhanced Raman
scattering (SERS). When it was observed that fluorescent molecules
showed enhanced emissions in the presence of this plasmonic effect
the field of MEF was born. A representation of the different
optical responses that occur when light is absorbed and scattered
by a metal nanoparticle can be seen. Due to above mechanism, the
plasmonic effect and related enhancement are near the surface
between 10 nm to 200 nm.
[0096] As noted above, in some embodiments, the method is done
without washing where, a washing step is a process that removes at
least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or a range between
any two of the values of the unbounded target analyte on the first
plate after binding step. Typically, the washing step contains
washing the plate with 1 times, 2 times, 3 times with a buffer.
[0097] In some embodiments, the second plate sample contact area
comprises a storage site containing detection agents that upon
contacting the sample, dissolves into the sample and diffuses in
the sample, wherein each capture agent, target analyte and
corresponding detection agent is capable of forming a capture
agent-target analyte-detection agent sandwich in a binding site of
the first plate.
Theoretical Analyze of Sensitivity of Wash-Free Homogeneous QMAX
Assay
[0098] Define final capture density (directly related to LoD or
sensitivity of the assay) of the target analyte (with label) on the
substrate (first plate) is d.sub.c;
[0099] Define the label density in the liquid is D.sub.L;
[0100] Define amplification factor is A;
[0101] Define amplification factor is uniform within L.sub.A of the
substrate;
[0102] Define liquid height is by X-Plate is L.sub.x
(L.sub.x>>L.sub.A);
[0103] Define the label signal intensity's standard deviation (sd)
of the liquid is .sigma.; Since signal from capture fluorophore
must be larger than (1+3.times.sd).times.background signal from
liquid, thus:
A .times. d c .gtoreq. ( 1 + 3 .times. .sigma. ) .times. ( D l
.times. L X + A .times. D l .times. L A ) ##EQU00001##
[0104] The smallest capture density (proportional to LoD)
detectable with this method is:
d c = ( 1 + 3 .times. .sigma. ) .times. ( L X + A .times. L A )
.times. D l A ##EQU00002##
[0105] Clearly, increase amplification factor (A) of substrate,
decrease QMAX thickness (L.sub.x) can improve the performance
(sensitivity) of wash-free homogeneous assay in QMAX card format.
But decrease the QMAX thickness might decrease the binding amount.
Thus there is a trade-off for the parameter of QMAX gap size or
liquid thickness.
Examples of QMAX Device for Nucleic Acid Hybridization Assay
[0106] FIG. 2 is an illustration of an exemplary nucleic acid
hybridization assay according to some embodiments of the present
invention.
Brief Summary of Assay Process
[0107] (A). Chip preparation: Capture probes were immobilized on
the substrate surface;
[0108] (B). Chip blocking: chips were blocked with blockers.
[0109] (C). Sample introduction: Biological fluids (whole blood,
plasma, serum, saliva, urine, sweat, etc.) containing targets of
interest, in the form of either free nucleic acids or cell/particle
contained nucleic acids, were added on the substrate surface;
[0110] (D) QMAX card closure and pressing: QMAX Card, with
micro-scale structure side facing down, were placed on top of the
chip and pressed. Necessary cell lysing reagents, protein
denaturing reagents, hybridization reagents and labeled detection
probe were dried on the side of QMAX Card with micro-scale
structures;
[0111] (E)-(F). Cell or particle lysing, target sequence capture
and detection: Dried reagents were dissolved in biological fluids.
If necessary, cells (or particles) were lysed by lysing reagents to
release target nucleic acid sequences. Released or free target
nucleic acid sequences were then captured by immobilized capture
probe on the substrate surface, and detected by labeled detection
probe through hybridization;
[0112] (G). Wash: QMAX Card was peeled off and the substrate
surface was washed with absorbing material containing suitable wash
solution;
[0113] (H). Signal detection: signals from labeled detection probes
were detected by detector.
Brief Experimental Procedures
[0114] As can be seen in FIG. 2(A), DNA oligonucleotide (capture
probe) with specific sequence that is complemented to region of
target nucleic acid sequence was coated on the surface of
substrate. DNA oligonucleotide, termed as "capture probe (1)" is
usually 10-50 bp in length, and 3' end modified to facilitate
coating on the substrate. Commonly used 3' end modifications
include but not limited to thiol, dithiol, amine, biotin, etc.
Substrates can be used for capture probe immobilization include but
not limited to gold surface, PMMA, PS, etc. Density of capture
probe coated on the substrate is critical to the accessibility of
the capture probe and thus affect the assay sensitivity. In one
application, single type capture probe, specific to a single target
nucleic acid sequence, can be immobilized on the substrate. In
another application, different types of capture probes, specific to
different target nucleic acid sequences or different regions of a
single target nucleic acid sequence, can be immobilized on the
substrate. Coating was ideally performed overnight at room
temperature, but can be shortened. After coating, the uncoated
capture probe was washed off using PBST buffer.
[0115] As can be seen in FIG. 2(B), the substrate surface was then
blocked with blocker solutions. Suitable blockers include but not
limited to small molecule blocks, such as 6-Mercapto-hexanol, or
protein blockers, such as bovine serum albumin, casein, milk
powder, etc. Blocking was performed for at least 30 min at room
temperature. The substrate surface was then washed with PBST and
ready to use.
[0116] As can be seen in FIG. 2(C), a biological sample is added
onto the surface of capture probe coated substrate. Biological
sample can be introduced by directly dropping on the substrate
surface or facilitated by transferring tools. Biological samples
that can be applied include but not limited to neat whole blood,
plasma, serum, urine, saliva, sweat, etc. Target of interest can be
in the form of free nucleic acid, nucleic acid and protein complex,
or within human cell, animal cell, plant cell, bacteria cells,
fungi cells, virus particles, etc. Target of interest includes but
not limited to linear nucleic acid, circular nucleic acid, single
strand nucleic acid, double strand nucleic acid, etc.
[0117] The term "nucleic acid" as used herein refers to any DNA or
RNA molecule, or a DNA/RNA hybrid, or mixtures of DNA and/or RNA.
The term "nucleic acid" therefore is intended to include but not
limited to genomic or chromosomal DNA, plasmid DNA, amplified DNA,
cDNA, total RNA, mRNA, miRNA, and small RNA. The term "nucleic
acid" is also intended to include natural DNA and/or RNA molecule,
or synthetic DNA and/or RNA molecule. In some embodiments,
cell-free nucleic acids are presence in the sample, as used herein
"cell-free" indicates nucleic acids are not contained in any
cellular structures. In some other embodiments, nucleic acids are
contained within cellular structures, which include but not limited
to human cells, animal cells, plant cells, bacterial cells, fungi
cells, and/or viral particles. Nucleic acids either in the form of
cell-free nucleic acids or within cellular structures or a
combination thereof, can be presence in the sample. In some further
embodiments, nucleic acids are purified before introduced onto the
inner surface of the first plate. In yet further embodiments,
nucleic acids can be within a complex associated with other
molecules, such as proteins and lipids.
[0118] As can be seen in FIG. 2(D), in some embodiments, necessary
reagents, including but limited to cell lysing reagents, protein
denaturing reagents, nucleic acid hybridization buffer, and labeled
detection probes, etc, were spotted or directly dried on the side
of X-plate (marked as QMAX card in some embodiments) with
micro-scale structures. After sample introduction, X-plate with
dried reagents (8) side facing down, was pressed on the sample and
to the substrate.
[0119] In some embodiments, cell lysing reagents include but not
limited to salts, detergents, enzymes, and other additives. The
term "salts" herein include but not limited to lithium salt (e.g.
lithium chloride), sodium salt (e.g. sodium chloride), potassium
(e.g. potassium chloride), Tris, and HEPES. The term "detergents"
herein can be ionic, including anionic and cationic, non-ionic or
zwitterionic. The term "ionic detergent" as used herein includes
any detergent which is partly or wholly in ionic form when
dissolved in water. Suitable anionic detergents include but not
limited to sodium dodecyl sulphate (SDS) or other alkali metal
alkylsulphate salts or similar detergents, sarkosyl, or
combinations thereof. The term "enzymes" herein include but not
limited to lysozyme, cellulase, and proteinase. In addition,
chelating agents including but not limited to EDTA, EGTA and other
polyamino carboxylic acids, and some reducing agents, such as
dithiotreitol (dTT), can also be included in cell lysing
reagents.
[0120] As can be seen in FIG. 2(E), after X-plate was pressed on
substrate, dried reagents were dissolved into biological fluids.
Cell lysing reagents facilitate breaking cell wall and cell
membranes to release the target nucleic acid analyte. Protein
denaturing reagents, such as SDS, denature nucleic acid associated
binding protein to release free nucleic acid. The composition of
dried hybridization reagent is critical to provide suitable salt
concentrations to maintain the strength of hybridization complex
and also reduce non-specific binding from biological samples. For
example, sodium chloride and sodium citrate were added to provide
ideal ionic strength in the hybridization buffer. Ficoll and
Polyvinylpyrrolidine (PVP) can be added to accelerate the
hybridization process. Bovine serum albumin is added to reduce
interference from biological samples. Labeled detection probe with
specific complementary sequence against target nucleic acid
sequence is used to detect target nucleic acid sequence through
hybridization.
[0121] As can be seen in Fig. (F), cell free target nucleic acid
sequences and/or released the target nucleic acid analytes were
captured by the capture probe through sequence specific
hybridization. Meanwhile, captured target nucleic acid sequences
were detected by labeled detection probe through sequence specific
hybridization. X-plate can be pressed on the substrate for certain
period of time. Experimental data indicated that after 2 min,
captured target nucleic acid sequence reached equilibrium.
[0122] As can be seen in FIG. 2(g), X-plate was peeled off from the
substrate. An absorbing material, such as sponge, containing
suitable wash buffer, preferably 5.times.SSC and 0.05% Tween 20,
was placed and softly pressed on the substrate surface. During
wash, cell debris, proteins, non-specific nucleic acid, etc. were
removed from the substrate surface.
[0123] In some embodiments, buffers with different ionic strengths
may be applied to increase signal to contrast ratio. Examples
include but not limited to, 0.1.times.SSC, 0.5.times.SSC,
1.times.SSC, 2.times.SSC, or 5.times.SSC. Washing step typically
contain washing the plate of 1 time, 2 times, or 3 times, or more
time. In some embodiments, each washing step may use the same type
of washing buffer. In some embodiments, different washing buffer
may be used in each washing step.
[0124] In some embodiments, 0.05% Tween 20 was used. In some
embodiments, other detergents may be used. As used herein, the term
"detergents" can be ionic, including anionic and cationic,
non-ionic or zwitterionic. The term "ionic detergent" as used
herein includes any detergent which is partly or wholly in ionic
form when dissolved in water. Suitable anionic detergents include
but not limited to sodium dodecyl sulphate (SDS) or other alkali
metal alkylsulphate salts or similar detergents, sarkosyl, or
combinations thereof.
[0125] As shown in FIG. 2(H), the absorbing material was peeled off
from the substrate. The signal intensity yielded from labeled
detection probe was measured by a suitable detector.
Examples for Nucleic Acid Hybridization Assays and Results
Example 1: miR21 Hybridization Assay in TE Buffer on M-Plate
Detected by IR800 Labelled Detection Probe Using 96 Well Plate
[0126] Assay details (referring to FIG. 3): [0127] 1 uM of
thiolated capture probe was coated on gold M-plate surface at room
temperature for overnight; [0128] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0129] Add 50 ul of miR21 target (diluted in TE
buffer) into each well, mixed with 50 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0130]
Hybridization for 2 h at room temperature; [0131] Rinse M-plate
with DNA washer (5.times.SSC+0.05% Tween 20) for 3 times; [0132]
Lump-sum signal measurement using Raman microscope.
[0133] Assay results (referring to FIG. 4): [0134] Correlation
between normalized signal intensities and the concentrations of
miR21 target in TE buffer was observed. [0135] Achieved a LoD of
510 fM miR21 target in TE buffer on M-plate using 2 h hybridization
protocol [0136] Achieved a dynamic range of 6 orders of
magnitude.
Example 2: miR21 Hybridization Assay in 10% Plasma on M-Plate
Detected by IR800 Labelled Detection Probe Using 96 Well Plate
[0137] Assay details (referring to FIG. 5): [0138] 1 uM of
thiolated capture probe was coated on gold M-plate surface at room
temperature for overnight; [0139] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0140] Add 50 ul of miR21 target (spiked in 10%
plasma) into each well, mixed with 50 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0141]
Hybridization for 2 h at room temperature; [0142] Rinse M-plate
with DNA washer (5.times.SSC+0.05% Tween 20) for 3 times; [0143]
Lump-sum signal measurement using Raman microscope.
[0144] Assay results (referring to FIG. 6): [0145] Correlation
between normalized signal intensities and the concentrations of
miR21 target in 10% plasma was observed. [0146] Achieved a LoD of
820 fM miR21 target in 10% plasma on M-plate using 2 h
hybridization protocol [0147] Achieved a dynamic range of 6 orders
of magnitude [0148] Assay sensitivity in 10% plasma is similar with
TE buffer, indicating non-significant interference in plasma
matrix
Example 3: miR21 Hybridization Assay in Neat Whole Blood on M-Plate
Detected by IR800 Labelled Detection Probe Using 96 Well Plate
[0149] Assay details (referring to FIG. 7): [0150] 1 uM of
thiolated capture probe was coated on gold M-plate surface at room
temperature for overnight; [0151] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0152] Add 50 ul of miR21 target (spiked in neat whole
blood) into each well, mixed with 50 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0153]
Hybridization for 2 h at room temperature; [0154] Rinse M-plate
with DNA washer (5.times.SSC+0.05% Tween 20) for 3 times; [0155]
Lump-sum signal measurement using Raman microscope.
[0156] Assay results (referring to FIG. 8): [0157] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed. [0158] Achieved a
LoD of 9.7 pM miR21 target in neat whole blood on M-plate using 2 h
hybridization protocol [0159] Achieved a dynamic range of 6 orders
of magnitude [0160] Assay sensitivity in neat whole blood is
comparable with TE buffer and 10% plasma, indicating robust assay
performance in the matrix of whole blood
Example 4: Time Course Study--miR21 Hybridization Assay in Neat
Whole Blood on Gold Thin Film Detected by IR800 Labelled Detection
Probe Using QMAX Card with 30 Um Spacers
[0161] Assay details (referring to FIG. 9): [0162] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0163] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0164] Drop 0.5 ul of 1 uM miR21 target (diluted in
neat whole blood) on chip surface, mixed with 0.5 ul of 1 uM IR800
labelled detection probe (diluted in H7140 hybridization buffer);
[0165] Pressed with QMAX card with 30 um spacers, and hybridization
for varied time at room temperature; [0166] Rinse chip surface with
DNA washer (5.times.SSC+0.05% Tween 20) for 3 times; [0167]
Lump-sum signal measurement using Raman microscope.
[0168] Assay results (referring to FIG. 10): [0169] When using
IR800 labelled detection probe in the miR21 hybridization assay,
and pressed by QMAX card with 30 um spacers, signal intensity is
higher at 2 min hybridization time compared to 1 min hybridization
time. [0170] After 2 min, signal intensity saturates [0171] Thus,
hybridization time of 2 min is used for further nucleic acid
hybridization assay involved using IR800 labelled detection probe
and QMAX card with 30 um spacers.
Example 5: Time Course Study--miR21 Hybridization Assay in Neat
Whole Blood on Gold Thin Film Detected by Streptavidin-40 nm Bead
Using QMAX Card with 30 Um Spacers
[0172] Assay details (referring to FIG. 11): [0173] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0174] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0175] Drop 0.5 ul of miR21 target (diluted in TE
buffer) on chip surface, mixed with 0.5 ul of 1 uM biotinylated
detection probe (diluted in H7140 hybridization buffer); [0176]
Pressed with QMAX card with 30 um spacers, and hybridization for 2
min at room temperature; [0177] Rinse chip surface with DNA washer
(5.times.SSC+0.05% Tween 20) for 3 times; [0178] Drop 1 ul of
streptavidin-40 nm bead (1:10 diluted in 4% BSA). [0179] Pressed
with QMAX card with 30 um spacers and wait for varied time at room
temperature; [0180] Rinse chip surface with DNA washer for 3 times;
[0181] Lump-sum signal measurement using Raman microscope
[0182] Assay results (referring to FIG. 12): [0183] When using
IR800 labelled detection probe in the miR21 hybridization assay,
and pressed by QMAX card with 30 um spacers, signal intensity is
higher at 2 min hybridization time compared to 1 min hybridization
time. [0184] Signal intensity peaked at 5 min [0185] Thus,
hybridization time of 5 min is used for further nucleic acid
hybridization assay involved using streptavidin-40 nm bead and QMAX
card with 30 um spacers.
Example 6: miR21 Hybridization Assay in TE Buffer on Gold Thin Film
Detected by IR800 Labelled Detection Probe Using QMAX Card with 30
Um Spacers
[0186] Assay details (referring to FIG. 13): [0187] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0188] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0189] Drop 0.5 ul of miR21 target (diluted in TE
buffer) on chip surface, mixed with 0.5 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0190]
Pressed with QMAX card with 30 um spacers, and hybridization for 2
min at room temperature; [0191] Rinse chip surface with DNA washer
(5.times.SSC+0.05% Tween 20) for 3 times; [0192] Lump-sum signal
measurement using Raman microscope.
[0193] Assay results (referring to FIG. 14): [0194] Correlation
between normalized signal intensities and the concentrations of
miR21 target in TE buffer was observed. [0195] Achieved a LoD of
861 pM miR21 target in TE buffer using QMAX card with 30 um spacers
under 2 min hybridization [0196] Achieved a dynamic range of 3
orders of magnitude
Example 7: miR21 Hybridization Assay in TE Buffer on Gold Thin Film
Detected by Biotinylated Detection Probe and Streptavidin-40 nm
Bead Using QMAX Card with 30 Um Spacers
[0197] Assay details (referring to FIG. 15): [0198] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0199] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0200] Drop 0.5 ul of miR21 target (diluted in TE
buffer) on chip surface, mixed with 0.5 ul of 1 uM biotinylated
detection probe (diluted in H7140 hybridization buffer); [0201]
Pressed with QMAX card with 30 um spacers, and hybridization for 2
min at room temperature; [0202] Rinse chip surface with DNA washer
(5.times.SSC+0.05% Tween 20) for 3 times; [0203] Drop 1 ul of
streptavidin-40 nm bead (1:10 diluted in 4% BSA). [0204] Pressed
with QMAX card with 30 um spacers and wait for 5 min at room
temperature; [0205] Rinse chip surface with DNA washer for 3 times;
[0206] Lump-sum signal measurement using Raman microscope.
[0207] Assay results (referring to FIG. 16): [0208] Correlation
between normalized signal intensities and the concentrations of
miR21 target in TE buffer was observed. [0209] Achieved a LoD of 28
nM miR21 target in TE buffer using QMAX card with 30 um spacers
under 2 min hybridization [0210] Achieved a dynamic range of 1
orders of magnitude
Example 8: miR21 Hybridization Assay in Neat Whole Blood on Gold
Thin Film Detected by IR800 Labelled Detection Probe Using QMAX
Card with 30 Um Spacers
[0211] Assay details (referring to FIG. 17): [0212] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0213] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0214] Drop 0.5 ul of miR21 target (diluted in neat
whole blood) on chip surface, mixed with 0.5 ul of 1 uM IR800
labelled detection probe (diluted in H7140 hybridization buffer);
[0215] Pressed with QMAX card with 30 um spacers, and hybridization
for 2 min at room temperature; [0216] Rinse chip surface with DNA
washer (5.times.SSC+0.05% Tween 20) for 3 times; [0217] Lump-sum
signal measurement using Raman microscope.
[0218] Assay results (referring to FIG. 18): [0219] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed. [0220] Achieved a
LoD of 8.41 pM miR21 target in neat whole blood using QMAX card
with 30 um spacers under 2 min hybridization [0221] Achieved a
dynamic range of 5 orders of magnitude [0222] Using QMAX card
significantly shorten the assay time (from 2 h to 2 min) with
almost the same sensitivity (9.7 pM compared to 8.41 pM) on gold
thin film
Example 9: miR21 Hybridization Assay in Neat Whole Blood on Gold
Thin Film Detected by Biotinylated Detection Probe and
Streptavidin-40 nm Bead Using QMAX Card with 30 Um Spacers
[0223] Assay details (referring to FIG. 19): [0224] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0225] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0226] Drop 0.5 ul of miR21 target (diluted in neat
whole blood) on chip surface, mixed with 0.5 ul of 1 uM
biotinylated detection probe (diluted in H7140 hybridization
buffer); [0227] Pressed with QMAX card with 30 um spacers, and
hybridization for 2 min at room temperature; [0228] Rinse chip
surface with DNA washer (5.times.SSC+0.05% Tween 20) for 3 times;
[0229] Drop 1 ul of streptavidin-40 nm bead (1:10 diluted in 4%
BSA). [0230] Pressed with QMAX card with 30 um spacers and wait for
5 min at room temperature; [0231] Rinse chip surface with DNA
washer for 3 times; [0232] Lump-sum signal measurement using Raman
microscope.
[0233] Assay results (referring to FIG. 20): [0234] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed. [0235] Achieved a
LoD of 8.8 nM miR21 target in neat whole blood using 2 min
hybridization protocol [0236] Achieved a dynamic range of 2 orders
of magnitude
Example 10: miR21 Hybridization Assay in Neat Whole Blood on Gold
Thin Film Detected by Biotinylated Detection Probe and
Streptavidin-40 nm Bead Using QMAX Card with 30 Um Spacers
[0237] Assay details (referring to FIG. 21): [0238] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0239] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0240] Drop 0.5 ul of miR21 target (diluted in neat
whole blood) on chip surface, mixed with 0.5 ul of 1 uM
biotinylated detection probe (diluted in H7140 hybridization
buffer); [0241] Pressed with QMAX card with 30 um spacers, and
hybridization for 2 min at room temperature; [0242] Rinse chip
surface with DNA washer (5.times.SSC+0.05% Tween 20) for 3 times;
[0243] Drop 1 ul of streptavidin-40 nm bead (1:10 diluted in 4%
BSA). [0244] Pressed with QMAX card with 30 um spacers and wait for
5 min at room temperature; [0245] Rinse chip surface with DNA
washer for 3 times; [0246] PIXelated measurement using inverted
microscope.
[0247] Assay results (referring to FIG. 22) [0248] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed. [0249] Achieved a
LoD of 87 pM miR21 target in neat whole blood using QMAX card with
30 um spacers under 2 min hybridization [0250] Achieved a dynamic
range of 2 orders of magnitude
Example 11: miR21 Hybridization Assay in Neat Whole Blood on Gold
Thin Film Detected by Biotinylated Detection Probe and
Streptavidin-Cy5 Using QMAX Card with 30 Um Spacers
[0251] Assay details (referring to FIG. 23): [0252] 1 uM of
thiolated capture probe was coated on gold thin film at room
temperature for overnight; [0253] Rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times; [0254] Drop 0.5 ul of miR21 target (diluted in neat
whole blood) on chip surface, mixed with 0.5 ul of 1 uM
biotinylated detection probe (diluted in H7140 hybridization
buffer); [0255] Pressed with QMAX card with 30 um spacers, and
hybridization for 2 min at room temperature; [0256] Rinse chip
surface with DNA washer (5.times.SSC+0.05% Tween 20) for 3 times;
[0257] Drop 1 ul of 500 ng/ml streptavidin-Cy5. [0258] Pressed with
QMAX card with 30 um spacers and wait for 2 min at room
temperature; [0259] Rinse chip surface with DNA washer for 3 times;
[0260] Lump-sum signal measurement using Raman microscope.
[0261] Assay results (referring to FIG. 24) [0262] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed. [0263] Achieved a
LoD of 68 pM miR21 target in neat whole blood using QMAX card with
30 um spacers under 2 min hybridization [0264] Achieved a dynamic
range of 4 orders of magnitude [0265] Assay sensitivity using
Streptavidin-Cy5 as detection label (68 pM) is not as good as using
IR800 as the label (8.41 pM)
Example 12: miR21 Hybridization Assay in Neat Whole Blood on
M-Plate Detected by IR800 Labelled Detection Probe Using QMAX Card
with 30 Um Spacers
[0266] Assay details (referring to FIG. 25): [0267] 1 uM of
thiolated capture probe was coated on M-plate at room temperature
for overnight; [0268] Rinsed with PBST for 3 times, and then
blocked with 50 uM MCH for 30 min, and then rinsed with PBST for 3
times; [0269] Drop 0.5 ul of miR21 target (diluted in neat whole
blood) on chip surface, mixed with 0.5 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0270]
Pressed with QMAX card with 30 um spacers, and hybridization for 2
min at room temperature; [0271] Rinse M-plate with DNA washer
(5.times.SSC+0.05% Tween 20) for 3 times; [0272] Lump-sum signal
measurement using Raman microscope.
[0273] Assay results (referring to FIG. 26) [0274] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed. [0275] Achieved a
LoD of 10 pM miR21 target in neat whole blood using QMAX card with
30 um spacers under 2 min hybridization [0276] Achieved a dynamic
range of 5 orders of magnitude [0277] Again, using QMAX card
significantly shorten the assay time (from 2 h to 2 min) with
almost the same sensitivity (10 pM compared to 8.41 pM) on gold
thin film [0278] However, using M-plate did not improve the assay
sensitivity probably due to the poor quality of M-plate.
Example 13: miR21 Hybridization Assay in Neat Whole Blood on
M-Plate Detected by IR800 Labelled Detection Probe Using QMAX Card
with 30 Um Spacers
[0279] Assay details (referring to FIG. 27): [0280] 1 uM of
thiolated capture probe was coated on M-plate at room temperature
for overnight; [0281] Rinsed with PBST for 3 times, and then
blocked with 50 uM MCH for 30 min, and then rinsed with PBST for 3
times; [0282] Drop 0.5 ul of miR21 target (diluted in neat whole
blood) on chip surface, mixed with 0.5 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0283]
Pressed with QMAX card with 30 um spacers, and hybridization for 2
min at room temperature; [0284] Rinse M-plate with DNA washer
(5.times.SSC+0.05% Tween 20) for 3 times; [0285] PIXelated
measurement using inverted microscope.
[0286] Assay results (referring to FIG. 28) [0287] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed [0288] Achieved a LoD
of 350 fM miR21 target in neat whole blood using QMAX card with 30
um spacers under 2 min hybridization using PIXelated measurement
method [0289] When measured by PIXelated method (350 fM), which is
approximately 2 orders more sensitive than Lump-sum measurement
method (10 pM) of the same assay
Example 14: miR21 Hybridization Assay in Neat Whole Blood on 30 Um
X-Well Detected by IR800 Labelled Detection Probe
[0290] Assay details (referring to FIG. 29): [0291] 1 uM of
thiolated capture probe was coated on X-Well at room temperature
for overnight; [0292] Rinsed with PBST for 3 times, and then
blocked with 50 uM MCH for 30 min, and then rinsed with PBST for 3
times; [0293] Drop 0.5 ul of miR21 target (diluted in neat whole
blood) on chip surface, mixed with 0.5 ul of 1 uM IR800 labelled
detection probe (diluted in H7140 hybridization buffer); [0294]
Pressed with PET film, and hybridization for 2 min at room
temperature; [0295] Rinse X-Well with DNA washer (5.times.SSC+0.05%
Tween 20) for 3 times; [0296] Lump-sum signal measurement using
Raman microscope.
[0297] Assay results (referring to FIG. 30) [0298] Correlation
between normalized signal intensities and the concentrations of
miR21 target in neat whole blood was observed [0299] Achieved a LoD
of 140 fM miR21 target in neat whole blood using QMAX card with 30
um spacers under 2 min hybridization [0300] Pressing with PET film
slightly improved the LoD (pressing LoD=140 pM v.s. without
pressing LoD=336 pM)
Example 15: Single Base Pair Differentiation in miR21 Hybridization
Assay in Neat Whole Blood on M-Plate Detected by IR800 Labelled
Detection Probe Using 96 Well Plate
[0301] Assay details (referring to FIGS. 31 and 32): [0302] 500 nM
of thiolated capture probe was coated on gold M-plate surface at
room temperature for overnight; [0303] Rinsed with PBST for 3
times, and then blocked with 50 uM MCH for 30 min, and then rinsed
with PBST for 3 times; [0304] Add 50 ul of 10 nM miR21 target
(diluted in neat whole blood) into each well, mixed with 50 ul of
100 nM IR800 labelled detection probe (diluted in H7140
hybridization buffer); [0305] Hybridization for 2 h at room
temperature; [0306] Rinse M-plate with DNA washer
(1.times.SSC+0.05% Tween 20) for 3 times; [0307] Lump-sum signal
measurement using Raman microscope.
[0308] Assay results (referring to FIGS. 33 and 34) [0309]
Perfectly matched miR21 target yielded the highest signal intensity
among all tested mismatched targets [0310] Impact of the mismatch
base pair on the signal intensity is location dependent. [0311]
Mismatch base pair locates at the center of the hybridization
region has more significant impact than those at the end of the
hybridization region [0312] Even only introducing a single
mismatched base pair at the end of hybridization region, it
resulted in approximately 3 fold decrease of signal intensity
Summary of Experiment Conditions
TABLE-US-00001 [0313] Substrate Example Sample Method Plate Volume
Label Measurement LoD 1 TE 96 well M-Plate 100 ul IR800 Lumpsum 510
nM 2 10% 96 well M-Plate 100 ul IR800 Lumpsum 820 fM Plasma 3 Whole
96 well M-Plate 100 ul IR800 Lumpsum 9.7 pM Blood 4 Whole 96 well
M-Plate 100 ul IR800 Lumpsum Blood 5 Whole 96 well M-Plate 100 ul
IR800 Lumpsum Blood 6 TE X-plate Gold thin 1 ul IR800 Lumpsum 861
pM film 7 TE X-plate Gold thin 1 ul 40 nm Lumpsum 28 nM film beads
8 Whole X-plate Gold thin 1 ul IR800 Lumpsum 8.41 pM Blood film 9
Whole X-plate Gold thin 1 ul 40 nm Lumpsum 8.8 nM Blood film beads
10 Whole X-plate Gold thin 1 ul 40 nm Pixelated 87 pM Blood film
beads 11 Whole X-plate Gold thin 1 ul Cy5 Lumpsum 68 pM Blood film
12 Whole X-well M-Plate 1 ul IR800 Lumpsum 10 pM Blood 13 Whole
X-plate M-Plate 1 ul IR800 Pixelated 350 fM Blood 14 Whole X-well
-- 1.5 ul IR800 Lumpsum 140 pM Blood 15 Whole X-well 1.5 ul IR800
Lumpsum Blood
[0314] In all experiments, the analyte is miR21 nucleic acid.
Examples of Present Invention
[0315] A1. A device for a nucleic acid hybridization assay,
comprising: [0316] a first plate, a second plate, and spacers,
wherein: [0317] i. the plates are movable relative to each other
into different configurations; [0318] ii. each plate respectively
comprises an inner surface that has a sample contact area for
contacting a sample that comprises a nucleic acid analyte, [0319]
iii. the spacers have a predetermined substantially uniform height,
[0320] iv. the first plate comprises a nucleic acid capture probe
that is coated on the inner surface of the first plate, and [0321]
v. the second plate comprises a nucleic acid detection probe that
is coated on the inner surface of the second plate; [0322] wherein
one of the configurations is an open configuration, in which: the
two plates are partially or entirely separated apart, the spacing
between the plates is not regulated by the spacers, and the sample
is deposited on one or both of the plates; [0323] wherein another
of the configurations is a closed configuration, which is
configured after the sample deposition in the open configuration,
and in the closed configuration: at least one spacer is between the
two plates, at least part of the sample deposited is compressed by
the plates into a layer of highly uniform thickness and is
substantially stagnant relative to the plates, wherein the uniform
thickness of the layer is confined by the inner surfaces of the two
plates and is regulated by the plates and the spacers; [0324]
wherein the capture probe is configured to bind complimentarily to
one part of the analyte and immobilize the analyte to the inner
surface of the first plate; and [0325] wherein the detection probe
is configured to diffuse into layer of uniform thickness and bind
complimentarily to another part the analyte to produce a detectable
signal. [0326] B1. A method of nucleic acid analysis, comprising:
[0327] (a) obtaining a liquid sample comprising a nucleic acid
analyte; [0328] (b) obtaining a device of any of paragraphs A1-A12;
wherein: [0329] (c) depositing the sample on one or both of the
plates when the plates are in an open configuration, [0330] (d)
after (c), bringing the two plates together and pressing the plates
into a closed configuration, [0331] (e) detecting and measuring the
signal from the layer of uniform thickness, thereby determining the
presence and/or amount of the nucleic acid analyte. [0332] A2. The
device of embodiment A1, wherein the first plate further comprises
blockers that are coated on the inner surface of the first plate.
[0333] A3. The device of any prior embodiments, wherein the first
plate and/or the second plate further comprise stabilizers that are
coated on the inner surface of the respective plate. [0334] A4. The
device of embodiment A3, wherein the stabilizer is selected from:
sugar, polymers, glycerol, and a mixture thereof. [0335] A5. The
device of embodiment A3, wherein the stabilizer is sucrose or
glucose. [0336] A6. The device of any prior embodiments, wherein
the capture probe is covalently bound to the inner surface of the
first plate. [0337] A7. The device of any prior embodiments,
wherein the capture probe is bound to the inner surface of the
first plate through a thiol bond. [0338] A8. The device of
embodiment A7, wherein the attachment reagent is protein A. [0339]
A9. The device of any prior embodiments, wherein the sample
comprises whole blood. [0340] A10. The device of any prior
embodiments, wherein the sample comprises blood serum. [0341] A11.
The device of any prior embodiments, wherein the spacers are fixed
on the inner surface of the second plate. [0342] A12. The device of
any prior embodiments, wherein the detection probe and/or the
capture probe have the length of 10-40 bp. [0343] B2. The method of
embodiment B1, wherein the first plate further comprises blockers
that are coated on the inner surface of the first plate. [0344] B3.
The method of any prior embodiments, wherein the first plate and/or
the second plate further comprise stabilizers that are coated on
the inner surface of the respective plate. [0345] B4. The method of
embodiment B3, wherein the stabilizer is selected from: sugar,
polymers, glycerol, and a mixture thereof. [0346] B5. The method of
embodiment B3, wherein the stabilizer is sucrose or glucose. [0347]
B6. The method of any prior embodiments, wherein the capture probe
is covalently bound to the inner surface of the first plate. [0348]
B7. The method of any prior embodiments, wherein the capture probe
is attached to the inner surface of the first plate by passive
absorption through hydrophobic interactions between inner surface
and non-polar residues on an attachment reagent that bound to the
capture probe. [0349] B8. The method of embodiment B7, wherein the
attachment reagent is protein A. [0350] B8. The method of any prior
embodiments, wherein the sample comprises whole blood. [0351] B9.
The method of any prior embodiments, wherein the sample comprises
blood plasma. [0352] B10. The method of any prior embodiments,
wherein the spacers are fixed on the inner surface of the second
plate. [0353] B11. The method of any prior embodiments, before step
(e) and after step (d), further comprising incubating the layer of
uniform thickness for a predetermined period of time. [0354] B12.
The method of embodiment B11, wherein the predetermined period of
time is equal to or longer than the time needed for the detection
probe to diffuse into the sample across the layer of uniform
thickness. [0355] B13. The method of any prior embodiments, wherein
the sample is deposited on the first plate. [0356] B14. The method
of any prior embodiments, before step (d) after step (c), further
comprising incubating the sample on the first plate for a
predetermined period of time. [0357] B15. The method of embodiment
B14, wherein the predetermined period of time is equal to or longer
than the time needed for the binding between the capture probe and
the analyte to reach an equilibrium. [0358] B16. The method of any
prior embodiments, before step (d) and after step (c), further
comprising washing the inner surface of the first plate. [0359]
B17. The method of any prior embodiments, before step (e) and after
step (d), further comprising switching the plates into the open
configuration and washing the inner surface of the first plate.
[0360] B18. The method of any prior embodiments, wherein the inner
surface of the first plate is washed with a washing solution
absorbed in a sponge. [0361] B19. The method of any prior
embodiments, wherein the washing is conducted by squeezing the
sponge to release the wash solution onto the inner surface of the
first plate and releasing the sponge to reabsorb the wash solution.
[0362] B20. The method of any prior embodiments, wherein the
washing improves the limit of detection (LOD) for the detectable
signal.
Related Documents
[0363] The present invention includes a variety of embodiments,
which can be combined in multiple ways as long as the various
components do not contradict one another. The embodiments should be
regarded as a single invention file: each filing has other filing
as the references and is also referenced in its entirety and for
all purpose, rather than as a discrete independent. These
embodiments include not only the disclosures in the current file,
but also the documents that are herein referenced, incorporated, or
to which priority is claimed.
(1) Definitions
[0364] The terms used in describing the devices, systems, and
methods herein disclosed are defined in the current application, or
in PCT Application (designating U.S.) Nos. PCT/US2016/045437 and
PCT/US0216/051775, which were respectively filed on Aug. 10, 2016
and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065,
which was filed on Feb. 7, 2017, U.S. Provisional Application No.
62/426,065, which was filed on Feb. 8, 2017, U.S. Provisional
Application No. 62/456,504, which was filed on Feb. 8, 2017, all of
which applications are incorporated herein in their entireties for
all purposes.
[0365] The terms "CROF Card (or card)", "COF Card", "QMAX-Card",
"Q-Card", "CROF device", "COF device", "QMAX-device", "CROF
plates", "COF plates", and "QMAX-plates" are interchangeable,
except that in some embodiments, the COF card does not comprise
spacers; and the terms refer to a device that comprises a first
plate and a second plate that are movable relative to each other
into different configurations (including an open configuration and
a closed configuration), and that comprises spacers (except some
embodiments of the COF card) that regulate the spacing between the
plates. The term "X-plate" refers to one of the two plates in a
CROF card, wherein the spacers are fixed to this plate. More
descriptions of the COF Card, CROF Card, and X-plate are given in
the provisional application Ser. No. 62/456,065, filed on Feb. 7,
2017, which is incorporated herein in its entirety for all
purposes.
(2) Q-Card, Spacer and Uniform Sample Thickness
[0366] The devices, systems, and methods herein disclosed can
include or use Q-cards, spacers, and uniform sample thickness
embodiments for sample detection, analysis, and quantification. In
some embodiments, the Q-card comprises spacers, which help to
render at least part of the sample into a layer of high uniformity.
The structure, material, function, variation and dimension of the
spacers, as well as the uniformity of the spacers and the sample
layer, are herein disclosed, or listed, described, and summarized
in PCT Application (designating U.S.) Nos. PCT/US2016/045437 and
PCT/US0216/051775, which were respectively filed on Aug. 10, 2016
and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065,
which was filed on Feb. 7, 2017, U.S. Provisional Application No.
62/426,065, which was filed on Feb. 8, 2017, U.S. Provisional
Application No. 62/456,504, which was filed on Feb. 8, 2017, all of
which applications are incorporated herein in their entireties for
all purposes.
(3) Hinges, Opening Notches, Recessed Edge and Sliders
[0367] The devices, systems, and methods herein disclosed can
include or use Q-cards for sample detection, analysis, and
quantification. In some embodiments, the Q-card comprises hinges,
notches, recesses, and sliders, which help to facilitate the
manipulation of the Q card and the measurement of the samples. The
structure, material, function, variation and dimension of the
hinges, notches, recesses, and sliders are herein disclosed, or
listed, described, and summarized in PCT Application (designating
U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/426,065, which was filed
on Feb. 8, 2017, U.S. Provisional Application No. 62/456,504, which
was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
(4) Q-Card, Sliders, and Smartphone Detection System
[0368] The devices, systems, and methods herein disclosed can
include or use Q-cards for sample detection, analysis, and
quantification. In some embodiments, the Q-cards are used together
with sliders that allow the card to be read by a smartphone
detection system. The structure, material, function, variation,
dimension and connection of the Q-card, the sliders, and the
smartphone detection system are herein disclosed, or listed,
described, and summarized in PCT Application (designating U.S.)
Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/426,065, which was filed
on Feb. 8, 2017, U.S. Provisional Application No. 62/456,504, which
was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
(5) Detection Methods
[0369] The devices, systems, and methods herein disclosed can
include or be used in various types of detection methods. The
detection methods are herein disclosed, or listed, described, and
summarized in PCT Application (designating U.S.) Nos.
PCT/US2016/045437 and PCT/US0216/051775, which were respectively
filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional
Application No. 62/456,065, which was filed on Feb. 7, 2017, U.S.
Provisional Application No. 62/426,065, which was filed on Feb. 8,
2017, U.S. Provisional Application No. 62/456,504, which was filed
on Feb. 8, 2017, all of which applications are incorporated herein
in their entireties for all purposes.
(6) Labels
[0370] The devices, systems, and methods herein disclosed can
employ various types of labels that are used for analytes
detection. The labels are herein disclosed, or listed, described,
and summarized in PCT Application (designating U.S.) Nos.
PCT/US2016/045437 and PCT/US0216/051775, which were respectively
filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional
Application No. 62/456,065, which was filed on Feb. 7, 2017, U.S.
Provisional Application No. 62/426,065, which was filed on Feb. 8,
2017, U.S. Provisional Application No. 62/456,504, which was filed
on Feb. 8, 2017, all of which applications are incorporated herein
in their entireties for all purposes.
(7) Analytes
[0371] The devices, systems, and methods herein disclosed can be
applied to manipulation and detection of various types of analytes
(including biomarkers). The analytes and are herein disclosed, or
listed, described, and summarized in PCT Application (designating
U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/426,065, which was filed
on Feb. 8, 2017, U.S. Provisional Application No. 62/456,504, which
was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
(8) Applications (Field and Samples)
[0372] The devices, systems, and methods herein disclosed can be
used for various applications (fields and samples). The
applications are herein disclosed, or listed, described, and
summarized in PCT Application (designating U.S.) Nos.
PCT/US2016/045437 and PCT/US0216/051775, which were respectively
filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional
Application No. 62/456,065, which was filed on Feb. 7, 2017, U.S.
Provisional Application No. 62/426,065, which was filed on Feb. 8,
2017, U.S. Provisional Application No. 62/456,504, which was filed
on Feb. 8, 2017, all of which applications are incorporated herein
in their entireties for all purposes.
(9) Cloud
[0373] The devices, systems, and methods herein disclosed can
employ cloud technology for data transfer, storage, and/or
analysis. The related cloud technologies are herein disclosed, or
listed, described, and summarized in PCT Application (designating
U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/426,065, which was filed
on Feb. 8, 2017, U.S. Provisional Application No. 62/456,504, which
was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
Flat Top of Pillar Spacers
[0374] In certain embodiments of the present invention, the spacers
are pillars that have a flat top and a foot fixed on one plate,
wherein the flat top has a smoothness with a small surface
variation, and the variation is less than 5, 10 nm, 20 nm, 30 nm,
50 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800
nm, 1000 nm, or in a range between any two of the values. A
preferred flat pillar top smoothness is that surface variation of
50 nm or less.
[0375] Furthermore, the surface variation is relative to the spacer
height and the ratio of the pillar flat top surface variation to
the spacer height is less than 0.5%, 1%, 3%, 5%, 7%, 10%, 15%, 20%,
30%, 40%, or in a range between any two of the values. A preferred
flat pillar top smoothness has a ratio of the pillar flat top
surface variation to the spacer height is less than 2%, 5%, or
10%.
Sidewall Angle of Pillar Spacers
[0376] In certain embodiments of the present invention, the spacers
are pillars that have a sidewall angle. In some embodiments, the
sidewall angle is less than 5 degree (measured from the normal of a
surface), 10 degree, 20 degree, 30 degree, 40 degree, 50 degree, 70
degree, or in a range between any two of the values. In a preferred
embodiment, the sidewall angle is less 5 degree, 10 degree, or 20
degree.
Formation of Uniform Thin Fluidic Layer by an Imprecise Force
Pressing
[0377] In certain embodiment of the present invention, a uniform
thin fluidic sample layer is formed by using a pressing with an
imprecise force. The term "imprecise pressing force" without adding
the details and then adding a definition for imprecise pressing
force. As used herein, the term "imprecise" in the context of a
force (e.g. "imprecise pressing force") refers to a force that (a)
has a magnitude that is not precisely known or precisely
predictable at the time the force is applied; (b) has a pressure in
the range of 0.01 kg/cm.sup.2 (centimeter square) to 100
kg/cm.sup.2, (c) varies in magnitude from one application of the
force to the next; and (d) the imprecision (i.e. the variation) of
the force in (a) and (c) is at least 20% of the total force that
actually is applied.
[0378] An imprecise force can be applied by human hand, for
example, e.g., by pinching an object together between a thumb and
index finger, or by pinching and rubbing an object together between
a thumb and index finger.
[0379] In some embodiments, the imprecise force by the hand
pressing has a pressure of 0.01 kg/cm2, 0.1 kg/cm2, 0.5 kg/cm2, 1
kg/cm2, 2 kg/cm2, kg/cm2, 5 kg/cm2, 10 kg/cm2, 20 kg/cm2, 30
kg/cm2, 40 kg/cm2, 50 kg/cm2, 60 kg/cm2, 100 kg/cm2, 150 kg/cm2,
200 kg/cm2, or a range between any two of the values; and a
preferred range of 0.1 kg/cm2 to 0.5 kg/cm2, 0.5 kg/cm2 to 1
kg/cm2, 1 kg/cm2 to 5 kg/cm2, 5 kg/cm2 to 10 kg/cm2 (Pressure).
Spacer Filling Factor.
[0380] The term "spacer filling factor" or "filling factor" refers
to the ratio of the spacer contact area to the total plate area",
wherein the spacer contact area refers, at a closed configuration,
the contact area that the spacer's top surface contacts to the
inner surface of a plate, and the total plate area refers the total
area of the inner surface of the plate that the flat top of the
spacers contact. Since there are two plates and each spacer has two
contact surfaces each contacting one plate, the filling fact is the
filling factor of the smallest.
[0381] For example, if the spacers are pillars with a flat top of a
square shape (10 um.times.10 um), a nearly uniform cross-section
and 2 um tall, and the spacers are periodic with a period of 100
um, then the filing factor of the spacer is 1%. If in the above
example, the foot of the pillar spacer is a square shape of 15
um.times.15 um, then the filling factor is still 1% by the
definition.
[0382] The method or device of any prior embodiment, wherein the
spacers have pillar shape and nearly uniform cross-section.
[0383] The method or device of any prior embodiment, wherein the
inter spacer distance (SD) is equal or less than about 120 um
(micrometer).
[0384] The method or device of any prior embodiment, wherein the
inter spacer distance (SD) is equal or less than about 100 um
(micrometer).
[0385] The method or device of any prior embodiment, wherein the
fourth power of the inter-spacer-distance (ISD) divided by the
thickness (h) and the Young's modulus (E) of the flexible plate
(ISD4/(hE)) is 5.times.106 um3/GPa or less.
[0386] The method or device of any prior embodiment, wherein the
fourth power of the inter-spacer-distance (ISD) divided by the
thickness (h) and the Young's modulus (E) of the flexible plate
(ISD4/(hE)) is 5.times.105 um3/GPa or less.
[0387] The method or device of any prior embodiment, wherein the
spacers have pillar shape, a substantially flat top surface, a
predetermined substantially uniform height, and a predetermined
constant inter-spacer distance that is at least about 2 times
larger than the size of the analyte, wherein the Young's modulus of
the spacers times the filling factor of the spacers is equal or
larger than 2 MPa, wherein the filling factor is the ratio of the
spacer contact area to the total plate area, and wherein, for each
spacer, the ratio of the lateral dimension of the spacer to its
height is at least 1 (one).
[0388] The method or device of any prior embodiment, wherein the
spacers have pillar shape, a substantially flat top surface, a
predetermined substantially uniform height, and a predetermined
constant inter-spacer distance that is at least about 2 times
larger than the size of the analyte, wherein the Young's modulus of
the spacers times the filling factor of the spacers is equal or
larger than 2 MPa, wherein the filling factor is the ratio of the
spacer contact area to the total plate area, and wherein, for each
spacer, the ratio of the lateral dimension of the spacer to its
height is at least 1 (one), wherein the fourth power of the
inter-spacer-distance (ISD) divided by the thickness (h) and the
Young's modulus (E) of the flexible plate (ISD4/(hE)) is
5.times.106 um3/GPa or less.
[0389] The device of any prior device embodiment, wherein the ratio
of the inter-spacing distance of the spacers to the average width
of the spacer is 2 or larger, and the filling factor of the spacers
multiplied by the Young's modulus of the spacers is 2 MPa or
larger.
[0390] The method or device of any prior embodiment, wherein the
analytes is proteins, peptides, nucleic acids, synthetic compounds,
or inorganic compounds.
[0391] The method or device of any prior embodiment, wherein the
sample is a biological sample selected from amniotic fluid, aqueous
humour, vitreous humour, blood (e.g., whole blood, fractionated
blood, plasma or serum), breast milk, cerebrospinal fluid (CSF),
cerumen (earwax), chyle, chime, endolymph, perilymph, feces,
breath, gastric acid, gastric juice, lymph, mucus (including nasal
drainage and phlegm), pericardial fluid, peritoneal fluid, pleural
fluid, pus, rheum, saliva, exhaled breath condensates, sebum,
semen, sputum, sweat, synovial fluid, tears, vomit, and urine.
[0392] The method or device of any prior embodiment, wherein the
spacers have a shape of pillars and a ratio of the width to the
height of the pillar is equal or larger than one.
[0393] The method of any prior embodiment, wherein the sample that
is deposited on one or both of the plates has an unknown
volume.
[0394] The method or device of any prior embodiment, wherein the
spacers have a shape of pillar, and the pillar has substantially
uniform cross-section.
[0395] The method or device of any prior embodiment, wherein the
samples is for the detection, purification and quantification of
chemical compounds or biomolecules that correlates with the stage
of certain diseases.
[0396] The method or device of any prior embodiment, wherein the
samples is related to infectious and parasitic disease, injuries,
cardiovascular disease, cancer, mental disorders, neuropsychiatric
disorders, pulmonary diseases, renal diseases, and other and
organic diseases.
[0397] The method or device of any prior embodiment, wherein the
samples is related to the detection, purification and
quantification of microorganism.
[0398] The method or device of any prior embodiment, wherein the
samples is related to virus, fungus and bacteria from environment,
e.g., water, soil, or biological samples.
[0399] The method or device of any prior embodiment, wherein the
samples is related to the detection, quantification of chemical
compounds or biological samples that pose hazard to food safety or
national security, e.g. toxic waste, anthrax.
[0400] The method or device of any prior embodiment, wherein the
samples is related to quantification of vital parameters in medical
or physiological monitor.
[0401] The method or device of any prior embodiment, wherein the
samples is related to glucose, blood, oxygen level, total blood
count.
[0402] The method or device of any prior embodiment, wherein the
samples is related to the detection and quantification of specific
DNA or RNA from biosamples.
[0403] The method or device of any prior embodiment, wherein the
samples is related to the sequencing and comparing of genetic
sequences in DNA in the chromosomes and mitochondria for genome
analysis.
[0404] The method or device of any prior embodiment, wherein the
samples is related to detect reaction products, e.g., during
synthesis or purification of pharmaceuticals.
[0405] The method or device of any prior embodiment, wherein the
samples is cells, tissues, bodily fluids, and stool.
[0406] The method or device of any prior embodiment, wherein the
sample is the sample in the fields of human, veterinary,
agriculture, foods, environments, and drug testing.
[0407] The method or device of any prior embodiment, wherein the
sample is a biological sample is selected from hair, finger nail,
ear wax, breath, connective tissue, muscle tissue, nervous tissue,
epithelial tissue, cartilage, cancerous sample, or bone.
[0408] The devices or methods of any prior embodiment, wherein the
inter-spacer distance is in the range of 5 um to 120 um. um
[0409] The devices or methods of any prior embodiment, wherein the
inter-spacer distance is in the range of 120 um to 200 um.
[0410] The device of any prior device embodiment, wherein the
flexible plates have a thickness in the range of 20 urn to 250 urn
and Young's modulus in the range 0.1 to 5 GPa.
[0411] The device of any prior device embodiment, wherein for a
flexible plate, the thickness of the flexible plate times the
Young's modulus of the flexible plate is in the range 60 to 750
GPa-um. The device of any prior device embodiment, wherein the
layer of uniform thickness sample is uniform over a lateral area
that is at least 1 mm2.
[0412] The device of any prior device embodiment, wherein the layer
of uniform thickness sample is uniform over a lateral area that is
at least 3 mm2.
[0413] The device of any prior device embodiment, wherein the layer
of uniform thickness sample is uniform over a lateral area that is
at least 5 mm2.
[0414] The device of any prior device embodiment, wherein the layer
of uniform thickness sample is uniform over a lateral area that is
at least 10 mm2.
[0415] The device of any prior device embodiment, wherein the layer
of uniform thickness sample is uniform over a lateral area that is
at least 20 mm2.
[0416] The device of any prior device embodiment, wherein the layer
of uniform thickness sample is uniform over a lateral area that is
in a range of 20 mm2 to 100 mm2.
[0417] The device of any prior device embodiment, wherein the layer
of uniform thickness sample has a thickness uniformity of up to
+/-5% or better.
[0418] The device of any prior device embodiment, wherein the layer
of uniform thickness sample has a thickness uniformity of up to
+/-10% or better.
[0419] The device of any prior device embodiment, wherein the layer
of uniform thickness sample has a thickness uniformity of up to
+/-20% or better.
[0420] The device of any prior device embodiment, wherein the layer
of uniform thickness sample has a thickness uniformity of up to
+/-30% or better.
Additional Notes
[0421] Further examples of inventive subject matter according to
the present disclosure are described in the following enumerated
paragraphs.
[0422] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise, e.g., when
the word "single" is used. For example, reference to "an analyte"
includes a single analyte and multiple analytes, reference to "a
capture agent" includes a single capture agent and multiple capture
agents, reference to "a detection agent" includes a single
detection agent and multiple detection agents, and reference to "an
agent" includes a single agent and multiple agents.
[0423] As used herein, the terms "adapted" and "configured" mean
that the element, component, or other subject matter is designed
and/or intended to perform a given function. Thus, the use of the
terms "adapted" and "configured" should not be construed to mean
that a given element, component, or other subject matter is simply
"capable of" performing a given function. Similarly, subject matter
that is recited as being configured to perform a particular
function may additionally or alternatively be described as being
operative to perform that function.
[0424] As used herein, the phrase, "for example," the phrase, "as
an example," and/or simply the terms "example" and "exemplary" when
used with reference to one or more components, features, details,
structures, embodiments, and/or methods according to the present
disclosure, are intended to convey that the described component,
feature, detail, structure, embodiment, and/or method is an
illustrative, non-exclusive example of components, features,
details, structures, embodiments, and/or methods according to the
present disclosure. Thus, the described component, feature, detail,
structure, embodiment, and/or method is not intended to be
limiting, required, or exclusive/exhaustive; and other components,
features, details, structures, embodiments, and/or methods,
including structurally and/or functionally similar and/or
equivalent components, features, details, structures, embodiments,
and/or methods, are also within the scope of the present
disclosure.
[0425] As used herein, the phrases "at least one of" and "one or
more of," in reference to a list of more than one entity, means any
one or more of the entity in the list of entity, and is not limited
to at least one of each and every entity specifically listed within
the list of entity. For example, "at least one of A and B" (or,
equivalently, "at least one of A or B," or, equivalently, "at least
one of A and/or B") may refer to A alone, B alone, or the
combination of A and B.
[0426] As used herein, the term "and/or" placed between a first
entity and a second entity means one of (1) the first entity, (2)
the second entity, and (3) the first entity and the second entity.
Multiple entity listed with "and/or" should be construed in the
same manner, i.e., "one or more" of the entity so conjoined. Other
entity may optionally be present other than the entity specifically
identified by the "and/or" clause, whether related or unrelated to
those entities specifically identified.
[0427] Where numerical ranges are mentioned herein, the invention
includes embodiments in which the endpoints are included,
embodiments in which both endpoints are excluded, and embodiments
in which one endpoint is included and the other is excluded. It
should be assumed that both endpoints are included unless indicated
otherwise. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art.
[0428] In the event that any patents, patent applications, or other
references are incorporated by reference herein and (1) define a
term in a manner that is inconsistent with and/or (2) are otherwise
inconsistent with, either the non-incorporated portion of the
present disclosure or any of the other incorporated references, the
non-incorporated portion of the present disclosure shall control,
and the term or incorporated disclosure therein shall only control
with respect to the reference in which the term is defined and/or
the incorporated disclosure was present originally.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0429] The following detailed description illustrates some
embodiments of the invention by way of example and not by way of
limitation. The section headings and any subtitles used herein are
for organizational purposes only and are not to be construed as
limiting the subject matter described in any way. The contents
under a section heading and/or subtitle are not limited to the
section heading and/or subtitle, but apply to the entire
description of the present invention.
[0430] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present claims are not entitled to antedate such publication by
virtue of prior invention. Further, the dates of publication
provided can be different from the actual publication dates which
can need to be independently confirmed.
[0431] The terms "CROF Card (or card)", "COF Card", "QMAX-Card",
"Q-Card", "CROF device", "COF device", "QMAX-device", "CROF
plates", "COF plates", and "QMAX-plates" are interchangeable,
except that in some embodiments, the COF card does not comprise
spacers; and the terms refer to a device that comprises a first
plate and a second plate that are movable relative to each other
into different configurations (including an open configuration and
a closed configuration), and that comprises spacers (except some
embodiments of the COF) that regulate the spacing between the
plates. The term "X-plate" refers to one of the two plates in a
CROF card, wherein the spacers are fixed to this plate. More
descriptions of the COF Card, CROF Card, and X-plate are described
in the provisional application Ser. No. 62/456,065, filed on Feb.
7, 2017 and U.S. Provisional Application No. 62/456,287, which was
filed on Feb. 8, 2017, and U.S. Provisional Application No.
62/456,504, which was filed on Feb. 8, 2017, all of which is
incorporated herein in their entirety for all purposes.
QMAX Device for Nucleic Acid Capturing for Sequencing
[0432] FIG. 1A-FIG. 1C is a schematic drawing for an exemplary
embodiment of a QMAX (Q: quantification; M: magnifying; A: adding
reagents; X: acceleration; also known as compressed regulated open
flow (CROF)) device that can be used for capturing nucleic acid for
sequencing. In FIG. 1A-FIG. 1C the QMAX device is in an open
configuration. FIG. 1A shows a QMAX comprising a first plate, a
second plate, and wells on the second plate. FIG. 1B shows a view
of well array on the first plate. FIG. 1C shows a view of pillar
array on the second plate.
[0433] A QMAX device for capturing nucleic acid for sequencing
comprising:
[0434] a first plate, a second plate, and wells, wherein [0435] (a)
the first and second plates are movable relative to each other into
different configurations, and have, on its respective surface, a
sample contact area for contacting a fluidic sample that contains a
target analyte; [0436] (b) the second plate has, in the sample
contact area, a plurality of the wells, wherein each well has (i) a
well depth of 50 um or less, (ii) a well volume substantially less
than that of the sample, and (iii) a binding site with capture
probes immobilized at the site, and the capture probes captures the
target probes;
[0437] wherein one of the configurations is an open configuration,
in which: the average spacing between the inner surface of the
first plate and the rim of the wells in the second plate is at
least 300 um and the sample is deposited on one or both of the
plates;
[0438] wherein another of the configurations is a close
configuration, which is the configuration after the sample is
deposited in the open configuration; in the closed configuration,
at least a part of the sample is inside the wells, and the average
spacing between the inner surface of the first plate and the rim of
the well in the second plate is less than 1/10 (one tenth) of the
average spacing in open configuration.
[0439] FIG. 36a-FIG. 36b is schematic drawings for exemplary
embodiments of wells on first plate of QMAX. FIG. 36a shows a view
of wells on first plate with (i) round shape with square lattice
(ii) rectangle shape with square lattice (iii) triangle shape with
hexagonal lattice (iv) round shape with aperiodicity. FIG. 36b
shows a view of well array on first plate with (i) no metal coating
(ii) metal coating on bottom of the well (iii) metal coating on
side wall of the well (iv) metal coating on both bottom and side
wall of the well.
[0440] In some embodiments, the well on the first plate has periods
(average well to well center distance) of 1 nm, 10 nm, 100 nm, 500
nm, 1 um, 5 um, 50 um, 500 um, 1 mm, or a range between any two of
the values; and a preferred range of 10 nm to 100 nm, 100 nm to 500
nm, 500 nm to 1 um, 1 um to 10 um, or 10 um to 50 um (Period).
[0441] In some embodiments, the well on the first plate has well
size (average length or diameter) of 1 nm, 10 nm, 100 nm, 500 nm, 1
um, 5 um, 50 um, 500 um, 1 mm, or a range between any two of the
values; and a preferred range of 10 nm to 100 nm, 100 nm to 500 nm,
500 nm to 1 um, 1 um to 10 um, or 10 um to 50 um (Size).
[0442] In some embodiments, the well on the first plate has depth
of 1 nm, 10 nm, 100 nm, 500 nm, 1 um, 5 um, 50 um, 500 um, 1 mm, or
a range between any two of the values; and a preferred range of 10
nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 10 um, or
10 um to 50 um (Depth). In some embodiments, well depth is 0,
meaning no well on the first plate.
[0443] In some embodiments, well is "pillar" instead, with pillar
height of 1 nm, 10 nm, 100 nm, 500 nm, 1 um, 5 um, 50 um, 500 um, 1
mm, or a range between any two of the values; and a preferred range
of 10 nm to 100 nm, 100 nm to 500 nm, 500 nm to 1 um, 1 um to 10
um, or 10 um to 50 um (pillar height).
[0444] In some embodiments, wells have (i) no metal coating (ii)
metal coating on bottom of the well (top of the pillar) (iii) metal
coating on side wall of the well (side of the pillar) (iv) metal
coating on both bottom and side wall of the well.
[0445] In some embodiments, the coating metal is gold, aluminum,
silver, copper, tin and/or their combinations.
[0446] In some embodiments, the well area ratio (ratio of the well
area to the total area of the surface) is 40% to 50%, 50% to 60%,
60% to 70%, 70% to 80%, 80% to 90%, 90% to 99% or a range between
any two of the values.
[0447] In some embodiments, the well numbers on the first plate is
larger than the nucleic acid probe numbers in the sample,
[0448] For example, total well number on the first plate is 1 to 2
times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000
times, 1000 to 10000 times of 600, If the nucleic acid probe
concentration is 1 fM with volume of 1 uL;
[0449] For example, total well number on the first plate is 1 to 2
times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000
times, 1000 to 10000 times of 600,000, If the nucleic acid probe
concentration is 1 pM with volume of 1 uL;
[0450] For example, total well number on the first plate is 1 to 2
times, 2 to 5 times, 5 to 10 times, 10 to 100 times, 100 to 1000
times, 1000 to 10000 times of 600,000,000, If the nucleic acid
probe concentration is 1 nM with volume of 1 uL; In some
embodiments, well number is in such way to achieve, after nucleic
acid capture step, most of the wells capture no more than 1 target
nucleic acid probe.
[0451] Table 1 shows one example of the DNA probe number in 1 uL
sample with concentration 1 fM to 100 pM.
[0452] Table 2 shows one example of the well number in 2 cm.times.2
cm area with well pitch 1 um to 1 mm.
[0453] For example, with well pitch 100 um, total well number on
first plate with size of 4 cm.sup.2 is 40000. If using such well
plate measure 1 fM DNA sample in 1 uL sample, which has 600 DNA
probes, statistically each well will have no more than one
molecule.
[0454] In some embodiments, the second plate is a X-Plate.
[0455] In some embodiments, the first plate can be any material
with flat or engineered solid surface. Examples for the first plate
include but are but not limited to: plastic, silicon, PMMA, gold
and glass. In some embodiments, the second plate can be any
material with flat or engineered solid surface. Examples for the
first plate include but are but not limited to: plastic, silicon,
PMMA, gold and glass.
[0456] In some embodiments, the first plate is made of
semiconductors including carbon, germanium, selenium, silicon,
gallium arsenide (GaAs), gallium nitride (GaN), indium phosphide
(InP), zinc selenide (ZnSe), and silicon carbide (SiC); metals
including gold, aluminum, silver, copper, tin and/or their
combinations.
[0457] In some embodiments, the surface of the first plate facing
the second plate is defined as the inner surface of the first
plate; the surface of the second plate that faces the first plate
are also defined as the inner surface of the second plate. In some
embodiments, the inner surfaces of the respective plates comprise a
sample contact area for contacting a sample that comprises nucleic
acid. The sample contact area can occupy part or the entirety of
the respective inner surface. As shown in FIG. 35a-FIG. 35c, the
second plate can comprises spacers that are fixed on the inner
surface of the second plate. It should be noted, however, that in
some embodiments the spacers are fixed on the inner surface of the
first plate and in other embodiments on the inner surfaces of both
the second plate and the first plate.
[0458] FIG. 35a-FIG. 35c shows a sectional view of the plates in an
open configuration, in which the first plate and second plate are
partially or entirely separated apart, allowing a sample to be
deposited on either one or both of the plates.
[0459] The first plate and second plate are moveable relative to
each other into different configuration. One of the configurations
is an open configuration, in which the two plates are partially or
entirely separated apart and the spacing between the plates are not
regulated by the spacers. FIG. 35a-FIG. 35c shows the plates in the
open configuration, in which a sample, can be added to first plate,
the second plate, or both of the plates. In some embodiments, the
inner surface of a respective plate comprises a sample contact
area, which occupies a part of the entirety of the inner surface.
In certain embodiments, the spacers are positioned within the
sample contact area. In some embodiments, the spacers are not fixed
to any one of the plates, but are mixed in the sample.
The Method of Using the QMAX Device for Nucleic Acid Capturing for
Sequencing
[0460] FIG. 37 is a flow chart showing the basic steps in an
exemplary process for conducting a nucleic acid probe capture step
in nucleic acid sequencing using the QMAX device.
[0461] A method of capturing nucleic acid probe for sequencing,
comprising: [0462] (a) preparing the first plate by coating capture
probes; [0463] (b) obtaining a device of claim 1; [0464] (c)
obtaining a sample that contains target nucleic acid; [0465] (d)
depositing the sample on one or both of the plates when the plates
are configured in an open configuration; in which: the average
spacing between the inner surface of the first plate and the rim of
the wells in the second plate is at least 300 um; [0466] (e) after
(d), moving the two plates of the device of claim 1 into a close
configuration, in which, at least a part of the sample is inside
the wells, and the average spacing between the inner surface of the
first plate and the rim of the well in the second plate is less
than 1/10 (one tenth) of the average spacing in open configuration;
[0467] (f) peeling off the second plate, and wash the first plate;
[0468] (g) following nucleic acid sequencing steps.
[0469] As used herein, the terms "nucleic acid" and "nucleotide"
are intended to be consistent with their use in the art and to
include naturally occurring species or functional analogs thereof.
Particularly useful functional analogs of nucleic acids are capable
of hybridizing to a nucleic acid in a sequence specific fashion or
capable of being used as a template for replication of a particular
nucleotide sequence. Naturally occurring nucleic acids generally
have a backbone containing phosphodiester bonds. An analog
structure can have an alternate backbone linkage including any of a
variety of those known in the art. Naturally occurring nucleic
acids generally have a deoxyribose sugar (e.g. found in
deoxyribonucleic acid (DNA)) or a ribose sugar (e.g. found in
ribonucleic acid (RNA)). A nucleic acid can contain nucleotides
having any of a variety of analogs of these sugar moieties that are
known in the art. A nucleic acid can include native or non-native
nucleotides. In this regard, a native deoxyribonucleic acid can
have one or more bases selected from the group consisting of
adenine, thymine, cytosine or guanine and a ribonucleic acid can
have one or more bases selected from the group consisting of
uracil, adenine, cytosine or guanine. Useful non-native bases that
can be included in a nucleic acid or nucleotide are known in the
art. The terms "probe" or "target," when used in reference to a
nucleic acid, are intended as semantic identifiers for the nucleic
acid in the context of a method or composition set forth herein and
does not necessarily limit the structure or function of the nucleic
acid beyond what is otherwise explicitly indicated. The terms
"probe" and "target" can be similarly applied to other analytes
such as proteins, small molecules, cells or the like.
[0470] As used herein, the term "capture probe" refers to nucleic
acid that hybridizes to nucleic acid having a complementary
sequence.
[0471] The term "complementary" as used herein refers to a
nucleotide sequence that base-pairs by hydrogen bonds to a target
nucleic acid of interest. In the canonical Watson-Crick base
pairing, adenine (A) forms a base pair with thymine (T), as does
guanine (G) with cytosine (C) in DNA. In RNA, thymine is replaced
by uracil (U). As such, A is complementary to T and G is
complementary to C. Typically, "complementary" refers to a
nucleotide sequence that is fully complementary to a target of
interest such that every nucleotide in the sequence is
complementary to every nucleotide in the target nucleic acid in the
corresponding positions. When a nucleotide sequence is not fully
complementary (100% complementary) to a non-target sequence but
still may base pair to the non-target sequence due to
complementarity of certain stretches of nucleotide sequence to the
non-target sequence, percent complementarily may be calculated to
assess the possibility of a non-specific (off-target) binding. In
general, a complementary of 50% or less does not lead to
non-specific binding. In addition, a complementary of 70% or less
may not lead to non-specific binding under stringent hybridization
conditions.
[0472] In some embodiments, the capture probe is attached to the
surface of the first plate. In certain embodiments, the capture
probe can immobilize the onto the inner surface of the first
plate.
[0473] As used herein, the term "coat," when used as a verb, is
intended to mean providing a layer or covering on a surface. At
least a portion of the surface can be provided with a layer or
cover. In some cases, the entire surface can be provided with a
layer or cover. In alternative cases, only a portion of the surface
will be provided with a layer or covering. The term "coat," when
used to describe the relationship between a surface and a material,
is intended to mean that the material is present as a layer or
cover on the surface. The material can seal the surface, for
example, preventing contact of liquid or gas with the surface.
However, the material need not form a seal. For example, the
material can be porous to liquid, gas, or one or more components
carried in a liquid or gas. Exemplary materials that can coat a
surface include, but are not limited to, a gel, polymer, organic
polymer, liquid, metal, a second surface, plastic, silica, or
gas.
[0474] As used herein, the term "attached" refers to the state of
two things being joined, fastened, adhered, connected or bound to
each other. For example, an analyte, such as a nucleic acid, can be
attached to a material, such as a gel or solid support, by a
covalent or non-covalent bond. A covalent bond is characterized by
the sharing of pairs of electrons between atoms. A non-covalent
bond is a chemical bond that does not involve the sharing of pairs
of electrons and can include, for example, hydrogen bonds, ionic
bonds, van der Waals forces, hydrophilic interactions and
hydrophobic interactions.
[0475] In several embodiments, primer nucleic acids that are
attached to the first plate can be used for capture and/or
amplification of template nucleic acids. The primers can be
universal primers that hybridize to a universal adapter sequence
that is attached to different target nucleic acids in a library
(i.e. each target nucleic acid includes a target region that
differs from other target nucleic acids in the library and several
target nucleic acids in the library have the same universal adapter
sequence). In some embodiments, a target nucleic acid can be
attached to gel material, and primers (whether in solution or also
attached to the gel) can be used to amplify the attached target
nucleic acid (i.e. the target nucleic acid can serve as a template
for amplification).
[0476] More particularly in step (a), in some embodiments, the
first plate comprises a capture probe that is coated fully or
partially on the inner surface of the first plate.
[0477] In some embodiments, the first plate comprises a capture
probe that is coated fully or partially inside the wells on the
first plate.
[0478] In some embodiments, the first plate comprises a capture
probe that is coated fully or partially on the metal inside the
wells on the first plate.
[0479] In some embodiments, the capture probes can be applied to
the surface by printing, spraying, soaking or any other method that
applies homogenous layer of capture probes. In certain embodiments,
the capture probes is dried on the first plate.
[0480] More particular in step (c), as used herein, the term
"library" refers to a collection of analytes having different
chemical compositions. Typically, the analytes in a library will be
different species having a common feature or characteristic of a
genera or class, but otherwise differing in some way. For example,
a library can include nucleic acid species that differ in
nucleotide sequence, but that are similar with respect to having a
sugar-phosphate backbone.
[0481] As used herein, the term "different", when used in reference
to nucleic acids, means that the nucleic acids have nucleotide
sequences that are not the same as each other. Two or more nucleic
acids can have nucleotide sequences that are different along their
entire length. Alternatively, two or more nucleic acids can have
nucleotide sequences that are different along a substantial portion
of their length. For example, two or more nucleic acids can have
target nucleotide sequence portions that are different for the two
or more molecules while also having a universal sequence portion
that is the same on the two or more molecules.
[0482] In some embodiments, the method of the present invention,
before step (f) and after step (e), further comprise incubating the
layer of uniform thickness for a predetermined period of time. In
certain embodiments, the predetermined period of time is equal to
or longer than the time needed for the target nucleic acids to
diffuse into the sample across the layer of uniform thickness.
[0483] In certain embodiments, the predetermined period of time is
equal to or longer than the time needed for the target nucleic
acids to diffuse into the sample across the layer of uniform
thickness and captured by capture probe.
[0484] In certain embodiments, the predetermined period of time is
equal to or longer than the time needed for the target nucleic
acids to diffuse into the sample across the layer of uniform
thickness, captured by capture probe and being amplified to produce
a clonal population of an individual nucleic acid in each of the
wells.
[0485] As used herein, the term "clonal population" refers to a
population of nucleic acids that is homogeneous with respect to a
particular nucleotide sequence. The homogenous sequence is
typically at least 10 nucleotides long, but can be even longer
including for example, at least 50, 100, 250, 500, 1000 or 2500
nucleotides long. A clonal population can be derived from a single
target nucleic acid or template nucleic acid. A clonal population
can include at least 2, 5, 10, 100, 1000 or more copies of a target
nucleotide sequence. The copies can be present in a single nucleic
acid molecule, for example, as a concatemer or the copies can be
present on separate nucleic acid molecules (i.e. a clonal
population can include at least 2, 5, 10, 100, 1000 or more nucleic
acid molecules having the same target nucleotide sequence).
Typically, all of the nucleic acids in a clonal population will
have the same nucleotide sequence. It will be understood that a
negligible number of contaminant nucleic acids or mutations (e.g.
due to amplification artifacts) can occur in a clonal population
without departing from clonality. Thus, a population can be at
least 80%, 90%, 95% or 99% clonal. In some cases 100% pure clonal
populations may be present.
[0486] A method set forth herein can use any of a variety of
amplification techniques. Exemplary techniques that can be used
include, but are not limited to, polymerase chain reaction (PCR),
rolling circle amplification (RCA), multiple displacement
amplification (MDA), or random prime amplification (RPA). In
particular embodiments, one or more primers used for amplification
can be attached to a gel material. In PCR embodiments, one or both
of the primers used for amplification can be attached to a gel
material. Formats that utilize two species of attached primer are
often referred to as bridge amplification because double stranded
amplicons form a bridge-like structure between the two attached
primers that flank the template sequence that has been copied.
Exemplary reagents and conditions that can be used for bridge
amplification are described, for example, in U.S. Pat. No.
5,641,658; U.S. Patent Publ. No. 2002/0055100; U.S. Pat. No.
7,115,400; U.S. Patent Publ. No. 2004/0096853; U.S. Patent Publ.
No. 2004/0002090; U.S. Patent Publ. No. 2007/0128624; and U.S.
Patent Publ. No. 2008/0009420, each of which is incorporated herein
by reference. PCR amplification can also be carried out with one of
the amplification primers attached to a gel material and the second
primer in solution. An exemplary format that uses a combination of
one solid phase-attached primer and a solution phase primer is
emulsion PCR as described, for example, in Dressman et al., Proc.
Natl. Acad. Sci. USA 100:8817-8822 (2003), WO 05/010145, or U.S.
Patent Publ. Nos. 2005/0130173 or 2005/0064460, each of which is
incorporated herein by reference. Emulsion PCR is illustrative of
the format and it will be understood that for purposes of the
methods set forth herein the use of an emulsion is optional and
indeed for several embodiments an emulsion is not used.
Furthermore, primers need not be attached directly to solid
supports as set forth in the ePCR references and can instead be
attached to a gel material as set forth herein. In some solid phase
PCR or bridge amplification formats, a target nucleic acid can be
attached to a gel material and used as a template for
amplification.
[0487] RCA techniques can be modified for use in a method of the
present disclosure. Exemplary components that can be used in an RCA
reaction and principles by which RCA produces amplicons are
described, for example, in Lizardi et al., Nat. Genet. 19:225-232
(1998) and US Pat. App. Pub. No. 2007/0099208 A1, each of which is
incorporated herein by reference. Primers used for RCA can be in
solution or attached to a gel material.
[0488] MDA techniques can be modified for use in a method of the
present disclosure. Some basic principles and useful conditions for
MDA are described, for example, in Dean et al., Proc Natl. Acad.
Sci. USA 99:5261-66 (2002); Lage et al., Genome Research 13:294-307
(2003); Walker et al., Molecular Methods for Virus Detection,
Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res.
20:1691-96 (1992); U.S. Pat. Nos. 5,455,166; 5,130,238; and
6,214,587, each of which is incorporated herein by reference.
Primers used for MDA can be in solution or attached to a gel
material.
[0489] In particular embodiments a combination of the
above-exemplified amplification techniques can be used. For
example, RCA and MDA can be used in a combination wherein RCA is
used to generate a concatameric amplicon in solution (e.g. using
solution-phase primers). The amplicon can then be used as a
template for MDA using primers that are attached to a gel material.
In this example, amplicons produced after the combined RCA and MDA
steps will be attached to the gel material. The amplicons will
generally contain concatameric repeats of a target nucleotide
sequence.
[0490] Amplification techniques, such as those exemplified above,
can be used to produce gel-containing features having multiple
copies of target nucleic acids. An individual feature, such as a
well, can have a clonal population of nucleotide sequences in the
form of a single molecule concatemer, such as those produced by
RCA, or in the form of many nucleic acid molecules having the same
sequence such as those produced by bridge PCR. Generally, the
nucleic acid(s) having several copies of the amplified target will
be attached to the gel material.
[0491] In certain embodiments, the predetermined period of time is
less than 10 seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute,
1.5 minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes,
7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20
minutes, 30 minutes, or 60 minutes, or in a range between any of
the two values.
[0492] In some embodiments, for the method of the present
invention, the sample is deposited on the first plate. In certain
embodiments, before step (e) after step (d), the sample is
incubated on the first plate for a predetermined period of time. In
certain embodiments, the predetermined period of time is equal to
or longer than the time needed for the binding between the capture
antibody and the analyte to reach an equilibrium. In certain
embodiments, the predetermined period of time is less than 10
seconds, 20 seconds, 30 seconds, 45 seconds, 1 minute, 1.5 minutes,
2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8
minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes,
or 60 minutes, or in a range between any of the two values.
[0493] In some embodiments, for the method of the present
invention, after step (e), the inner surface of the first plate can
be washed to remove unbound molecules. For this approach, washing
is conducted before switch the plates into the closed
configuration. In some embodiments, for the method of the present
invention, before step (e) and after step (d), before step (f) and
after step (e), the plates can be switched into the open
configuration (e.g. by removing the second plate) and the inner
surface of the first plate can be washed. For this approach,
washing is conducted before switch the plates into the closed
configuration. In certain embodiments, such a step reduces
non-specific binding and reduce signal noise. In certain
embodiments, each of the wash step includes only one or multiple
washes. In some embodiments, both of the washing steps are
conducted. In some embodiments, only one of the washing steps is
conducted.
[0494] In some embodiments, the inner surface can be washed with
washing solution absorbed in a sponge. In some embodiments, the
washing is conducted by squeezing the sponge to release the wash
solution onto the inner surface of the first plate and releasing
the sponge to reabsorb the wash solution. In some embodiments, the
washing improves the limit of detection (LOD) for the detectable
signal.
[0495] Following the step (f), nucleic acids captured on the first
plate can be used in many biological applications, including
sequencing procedure, such as a sequencing-by-synthesis (SBS)
technique. Briefly, SBS can be initiated by contacting the target
nucleic acids with one or more labeled nucleotides, DNA polymerase,
etc. Those features where a primer is extended using the target
nucleic acid as template will incorporate a labeled nucleotide that
can be detected. Optionally, the labeled nucleotides can further
include a reversible termination property that terminates further
primer extension once a nucleotide has been added to a primer. For
example, a nucleotide analog having a reversible terminator moiety
can be added to a primer such that subsequent extension cannot
occur until a deblocking agent is delivered to remove the moiety.
Thus, for embodiments that use reversible termination, a deblocking
reagent can be delivered to the flow cell (before or after
detection occurs). Washes can be carried out between the various
delivery steps. The cycle can then be repeated n times to extend
the primer by n nucleotides, thereby detecting a sequence of length
n. Exemplary SBS procedures, fluidic systems and detection
platforms that can be readily adapted for use with an array
produced by the methods of the present disclosure are described,
for example, in Bentley et al., Nature 456:53-59 (2008), WO
04/018497; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,057,026;
7,329,492; 7,211,414; 7,315,019 or 7,405,281, and US Pat. App. Pub.
No. 2008/0108082 A1, each of which is incorporated herein by
reference.
[0496] Other sequencing procedures that use cyclic reactions can be
used, such as pyrosequencing. Pyrosequencing detects the release of
inorganic pyrophosphate (PPi) as particular nucleotides are
incorporated into a nascent nucleic acid strand (Ronaghi, et al.,
Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res.
11 (1), 3-11 (2001); Ronaghi et al. Science 281 (5375), 363 (1998);
U.S. Pat. Nos. 6,210,891; 6,258,568 and 6,274,320, each of which is
incorporated herein by reference). In pyrosequencing, released PPi
can be detected by being converted to adenosine triphosphate (ATP)
by ATP sulfurylase, and the resulting ATP can be detected via
luciferase-produced photons. Thus, the sequencing reaction can be
monitored via a luminescence detection system. Excitation radiation
sources used for fluorescence based detection systems are not
necessary for pyrosequencing procedures. Useful fluidic systems,
detectors and procedures that can be used for application of
pyrosequencing to arrays of the present disclosure are described,
for example, in WIPO Pat. App. Ser. No. PCT/US11/57111, US Pat.
App. Pub. No. 2005/0191698 A1, U.S. Pat. Nos. 7,595,883, and
7,244,559, each of which is incorporated herein by reference.
[0497] Sequencing-by-ligation reactions are also useful including,
for example, those described in Shendure et al. Science
309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each
of which is incorporated herein by reference. Some embodiments can
include sequencing-by-hybridization procedures as described, for
example, in Bains et al., Journal of Theoretical Biology 135 (3),
303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58
(1998); Fodor et al., Science 251 (4995), 767-773 (1995); and WO
1989/10977, each of which is incorporated herein by reference. In
both sequencing-by-ligation and sequencing-by-hybridization
procedures, nucleic acids that are present in gel-containing wells
(or other concave features) are subjected to repeated cycles of
oligonucleotide delivery and detection. Fluidic systems for SBS
methods as set forth herein, or in references cited herein, can be
readily adapted for delivery of reagents for sequencing-by-ligation
or sequencing-by-hybridization procedures. Typically, the
oligonucleotides are fluorescently labeled and can be detected
using fluorescence detectors similar to those described with regard
to SBS procedures herein or in references cited herein.
[0498] Some embodiments can utilize methods involving the real-time
monitoring of DNA polymerase activity. For example, nucleotide
incorporations can be detected through fluorescence resonance
energy transfer (FRET) interactions between a fluorophore-bearing
polymerase and .gamma.-phosphate-labeled nucleotides, or with
zeromode waveguides. Techniques and reagents for FRET-based
sequencing are described, for example, in Levene et al. Science
299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028
(2008); Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181
(2008), the disclosures of which are incorporated herein by
reference.
[0499] Some SBS embodiments include detection of a proton released
upon incorporation of a nucleotide into an extension product. For
example, sequencing based on detection of released protons can use
an electrical detector and associated techniques that are
commercially available from Ion Torrent (Guilford, Conn., a Life
Technologies subsidiary) or sequencing methods and systems
described in US Pat. App. Pub. Nos. 2009/0026082 A1; 2009/0127589
A1; 2010/0137143 A1; or 2010/0282617 A1, each of which is
incorporated herein by reference. In particular embodiments, the
electrical detectors that are used to detect the released protons
can be modified to include wells and the wells can contain gel
material as set forth herein.
[0500] In some embodiments of QMAX, the sample contact area of one
or both of the plates comprises a compressed open flow monitoring
surface structures (MSS) that are configured to monitoring how much
flow has occurred after COF. For examples, the MSS comprises, in
some embodiments, shallow square array, which will cause friction
to the components (e.g. blood cells in a blood) in a sample. By
checking the distributions of some components of a sample, one can
obtain information related to a flow, under a COF, of the sample
and its components.
[0501] The depth of the MSS can be 1/1000, 1/100, 1/100, 1/5, 1/2
of the spacer height or in a range of any two values, and in either
protrusion or well form.
Examples of Present Invention
[0502] FIG. 38 shows an example of first plate preparation step for
nucleic acid sequencing.
[0503] The first plate in this example is square well with size of
3 um by 3 um, period of 5 um, depth of 1 um fabricated on 1 mm
thick acrylic substrate. On the bottom of each well has 100 nm gold
and 1 nm titanium deposited. In this device, each 1 mm.sup.2 area
first plate has 40,000 wells. The fabrication of this nanostructure
by large area nanofabrication methods including nanoimprint
lithography, reactive ion etching (RIE), E-beam evaporation and
others.
[0504] 1 uM of thiolated capture probe was selected coated on gold
on well bottom at room temperature for overnight;
[0505] Then the first plate was rinsed with PBST for 3 times, and
then blocked with 50 uM MCH for 30 min, and then rinsed with PBST
for 3 times;
[0506] FIG. 39 is a schematic drawing for an exemplary embodiment
of a QMAX device in a closed configuration for capturing target
nucleic acid.
[0507] Add 1 ul of target probe (concentration is much less than 1
pM diluted in TE buffer, which is to make sure the number of total
nucleic acid probes in sample is much less than the well numbers on
the first plate) onto first plate in open configuration;
[0508] In some embodiments, the target probe is conjugated a label
to facilize the following measurement and evaluation of binding
probes. The label is a light-emitting label or an optical
detectable label, directly or indirectly, either prior to or after
it is bound to said capture agent. The label is label with signal
of Raman scattering, chromaticity, luminescence, fluorescence,
electroluminescence, chemiluminescence, and/or
electrochemiluminescence. As used herein, the term "light-emitting
label" refers to a label that can emit light when under an external
excitation. This can be luminescence. Fluorescent labels (which
include dye molecules or quantum dots), and luminescent labels
(e.g., electro- or chemi-luminescent labels) are types of
light-emitting label. The external excitation is light (photons)
for fluorescence, electrical current for electroluminescence and
chemical reaction for chemi-luminscence. An external excitation can
be a combination of the above. The phrase "labeled analyte" refers
to an analyte that is detectably labeled with a light emitting
label such that the analyte can be detected by assessing the
presence of the label. A labeled analyte may be labeled directly
(i.e., the analyte itself may be directly conjugated to a label,
e.g., via a strong bond, e.g., a covalent or non-covalent bond), or
a labeled analyte may be labeled indirectly (i.e., the analyte is
bound by a secondary capture agent that is directly labeled).
[0509] Press the second plate on top of the liquid by hand and QMAX
is in close configuration; Second plate used here is a X-Plate with
30.times.40 um pillar size, 80 um inter spacing distance, 30 um
pillar height on 175 um PMMA substrate.
[0510] Incubate the QMAX device for 1 min; peel off the second
plate;
[0511] Rinse the first plate with sponge, which contains DNA washer
(5.times.SSC+0.05% Tween 20) for 3 times;
[0512] The first plate is ready for following nucleic acid
sequencing steps.
Examples for Results
[0513] FIG. 40 shows representative a time course study for
capturing target DNA with (a) QMAX using 1 pM concentration 1 uL
target nucleic acid sample; (b) normal 96 microwell plate using 10
fM concentration 100 uL nucleic acid sample; (c) normal 96
microwell plate using 1 pM concentration 100 uL target nucleic acid
sample. The x-axis is the incubation time. The y-axis is the
fluorescence signal from IR-800 dye conjugated on captured nucleic
acid. The experiment process follows the flow chart of FIG. 37.
[0514] Clearly, QMAX device has the fastest incubation saturation
time (around 1 min) and requires smallest sample volume (1 uL).
While incubating first plate in normal 96 microwell, with low
concertation (10 fM) 100 uL sample, capturing process is not
saturated within 120 min, and first plate has much lower signal;
While incubating first plate in normal 96 microwell, with same high
concertation (1 pM) 100 uL sample, capturing process is not
saturated as fast as QMAX device and requires 100 times sample
volume than QMAX device.
[0515] As demonstrated by the examples, in some embodiments, the
present invention provides a platform for capturing nucleic acid
for sequencing, that is fast, simple, portable and only requires as
little as 1 .mu.L or less of sample. With the current invention,
nucleic acid capturing can be performed in a shallow enclosed space
with designated parameters so that the sample volume and capturing
time can be accurately controlled. In some embodiments, Brownian
motion of molecules is restricted in the shallow space so that
equilibrium of molecule binding can be reached faster. This
platform can be adapted for any nucleic acid sequencing that are
performed in traditional micro titter plate and thus have broad
applications.
Additional Examples
[0516] In the device or method herein described, the spacers can
have a height of 100 nm, 1 um, 5 um, 10 um, 20 um, 50 um, 500 um, 1
mm, or a range between any two of the values; and a preferred range
of 500 nm to 1 um, 1 um to 10 um, or 10 um to 30 um, 30 um to 50
um.
[0517] In the device or method herein described, the spacers can
have a height of 1 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 0.1 s for protein,
0.1 s for nucleic acid (20 bp).
[0518] In the device or method herein described, the spacers can
have a height of 2 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 0.3 s for protein,
0.1 s for nucleic acid (20 bp).
[0519] In the device or method herein described, the spacers can
have a height of 5 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 2 s for protein, 1 s
for nucleic acid (20 bp).
[0520] In the device or method herein described, the spacers can
have a height of 10 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 10 s for protein, 5
s for nucleic acid (20 bp).
[0521] In the device or method herein described, the spacers can
have a height of 30 um or less, thereby the saturation incubation
time (or saturation binding time) is less than for 60 s protein, 30
s for nucleic acid (20 bp).
[0522] In the device or method herein described, the spacers can
have a height of 50 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 2 min for protein, 1
min for nucleic acid (20 bp).
[0523] In the device or method herein described, the spacers can
have a height of 100 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 10 min for protein,
5 min for nucleic acid (20 bp).
[0524] In the device or method herein described, the sample can be
incubated for a saturation binding time. In some embodiments, the
saturation binding time is equal to or longer than the time that it
takes for the target entity to diffuse across the thickness of the
uniform thickness layer at the closed configuration. In some
embodiments, the saturation binding time is equal to or longer than
the time that it takes for the target entity to diffuse across the
thickness of the uniform thickness layer at the closed
configuration and to bind to the binding site. In some embodiments,
the saturation binding time is significantly shorter than the time
that it takes the target entity to laterally diffuse across the
minimum lateral dimension of the binding site. In some embodiments,
the saturation binding time is wherein at the end of the
incubation, the majority of the target entity bound to the binding
site is from a relevant volume of the sample. In some embodiments,
the incubation allows the target entity to bind to the binding
site, and wherein the relevant volume is a portion of the sample
that is above the binding site at the closed configuration.
[0525] In certain embodiments, the incubation time is in the range
of 1 seconds to 10 seconds. In certain embodiments, the incubation
time is in the range of 10 seconds to 30 seconds. In certain
embodiments, the incubation time is in the range of 30 seconds to
60 seconds. In certain embodiments, the incubation time is in the
range of 1 minutes to 5 minutes. In certain embodiments, the
incubation time is in the range of 5 minutes to 10 minutes. In
certain embodiments, the incubation time is in the range of 10
minutes to 30 minutes.
Present Embodiments
[0526] A method for performing a homogeneous nucleic acid detection
assay comprising:
[0527] (a) obtaining a QMAX device comprising a first plate and a
second plate, wherein [0528] the first plate and the second plate,
each comprises a sample contacting area for contacting a sample
that contains one or more target nucleic acids; [0529] the first
plate comprises, on its sample contacting area, a binding site that
comprises: [0530] (i) surface amplification surface; and [0531]
(ii) target-specific nucleic acid probes that are immobilized on
said amplification surface and that specifically binds to a part of
the target nucleic acid; and the second plate comprises a sample
contact area comprising a reagent storage site that comprises
target-specific nucleic acid detection agents that specifically
binds to another part of the target nucleic acid;
[0532] (b) depositing the sample on one or both of the plates when
the plates are in an open configuration;
[0533] (c) closing the plates to a closed configuration; and.
[0534] (d), after (c), while the plates remain in the closed
configuration and without any washing step, detecting the target
nucleic acid by reading the sample contact area with a reading
device to produce an image of signals; [0535] wherein: (i) the
thickness of the sample in the closed configuration, (ii) the
concentration of labels dissolved in the sample in the closed
configuration, and (iii) the amplification factor of the
proximity-dependent amplification surface are configured such that
labels that are indirectly bound to the nucleic acid probes via a
target nucleic acid are visible without washing away any biological
materials or labels that are not bound to the surface amplification
surface;
[0536] wherein one of the configurations is an open configuration,
in which the average spacing between the inner surfaces of the two
plates is at least 200 um; and
[0537] wherein another of the configurations is a close
configuration, in which, at least part of the sample is between the
two plates and the average spacing between the inner surfaces of
the plates is less than 200 um.
[0538] A device for analyzing a homogenous sample comprising:
[0539] a first plate, a second plate, and a binding site,
wherein
[0540] (a) the first and second plates are movable relative to each
other into different configurations, and have, on its respective
surface, a sample contact area for contacting a sample that
contains a target analyte,
[0541] (b) the sample contact area on the first plate has a binding
site that comprises: [0542] (i) proximity-dependent signal
amplification layer, and [0543] (ii) target-specific nucleic acid
probes that are attached to said proximity-dependent signal
amplification layer that bind to part of a target nucleic acid;
[0544] (c) the sample contact area on the second plate comprising a
reagent storage site that comprises target-specific nucleic acid
detection agents that bind to another part of the target nucleic
acid;
[0545] wherein one of the configurations is an open
configuration;
[0546] wherein another of the configurations is a close
configuration, in which, at least part of the sample is between the
two plates; and
[0547] wherein the thickness of the sample in the closed
configuration, the concentration of the labels dissolved in the
sample in the closed configuration, and the amplification factor of
the proximity-dependent signal amplification layer are configured
such that any the labels that are indirectly bound to the
target-specific nucleic acid probes are visible without washing
away of the unbound labels.
[0548] An apparatus comprising a thermal cycler and a device of
embodiment 2.
[0549] An apparatus comprising a thermal cycler, a device of
embodiment 2, and a reader for real-time PCR.
[0550] A method for rapid nucleic acid detection assay
comprising:
[0551] (a) obtaining a QMAX device comprising a first plate and a
second plate, wherein [0552] the first plate and the second plate,
each comprises a sample contacting area for contacting a sample
that contains one or more target nucleic acids; [0553] the first
plate comprises, on its sample contacting area, a binding site that
comprises target-specific nucleic acid probes that are immobilized
on the site and that specifically binds to part of the target
nucleic acid; and [0554] the second plate comprises a sample
contact area comprising a reagent storage site that comprises
target-specific nucleic acid detection agents that specifically
binds to another part of the target nucleic acid;
[0555] (b) depositing the sample on one or both of the plates when
the plates are in an open configuration;
[0556] (c) closing the plates to a closed configuration for
incubation for a period of time; and.
[0557] (d) opening the plates and pressing the plate again a
washing sponge that has washing solution for a period of time and
then releasing the washing sponge;
[0558] (e), after (d), reading the sample contact area with a
reading device to produce an image of signals; [0559] wherein: (i)
the thickness of the sample in the closed configuration, (ii) the
concentration of labels dissolved in the sample in the closed
configuration, and (iii) the amplification factor of the
proximity-dependent amplification surface are configured such that
labels that are indirectly bound to the nucleic acid probes via a
target nucleic acid are visible without washing away any biological
materials or labels that are not bound to the proximity-dependent
amplification surface;
[0560] wherein one of the configurations is an open configuration,
in which the average spacing between the inner surfaces of the two
plates is at least 200 um; and
[0561] wherein another of the configurations is a close
configuration, in which, at least part of the sample is between the
two plates and the average spacing between the inner surfaces of
the plates is less than 200 um.
[0562] The device, apparatus or method of any prior embodiment,
wherein the spacing between the first plate and the second plate in
the closed configuration is configured to make saturation binding
time of the target analyte to the capture agents 300 sec or
less.
[0563] The device, apparatus or method of any prior embodiment,
wherein the spacing between the first plate and the second plate in
the closed configuration is configured to make saturation binding
time of the target analyte to the capture agents 300 sec or
less.
[0564] The device, apparatus or method of any prior embodiment,
wherein the spacing between the first plate and the second plate in
the closed configuration is configured to make saturation binding
time of the target analyte to the capture agents 60 sec or
less.
[0565] The device, apparatus or method of any prior embodiment,
wherein the target nucleic acid is a DNA or RNA, including genomic
DNA, cfDNA, cDNA ctDNA, mRNA and miRNA.
[0566] The device, apparatus or method of any prior embodiment,
wherein the time from step (b) to obtaining a result is less than
10 min.
[0567] The device, apparatus or method of any prior embodiment,
wherein the thickness of the sample in the closed configuration,
the concentration of the labels dissolved in the sample in the
closed configuration, and the amplification factor of the surface
amplification layer are configured such that any the labels that
are bound directly or indirectly to the probles are visible in the
closed configuration without washing away of the unbound
labels.
[0568] The device, apparatus or method of any prior embodiment,
wherein he labels bound to the proximity-dependent amplification
surface are visible in less than 60 seconds.
[0569] The device, apparatus or method of any prior embodiment,
wherein, wherein the labels bound to the proximity-dependent
amplification surface are visible in less than 60 seconds.
[0570] The device, apparatus or method of any prior embodiment,
wherein, wherein the storage site is approximately above the
binding site on the first plate in the closed configuration.
[0571] The device, apparatus or method of any prior embodiment,
wherein, wherein the target-specific nucleic acid probes and the
target-specific nucleic acid detection agents form a sandwich that
comprises the label.
[0572] The device, apparatus or method of any prior embodiment,
wherein, wherein the signals are read without using a wash step to
remove any biological materials or labels that are not bound to the
amplification surface.
[0573] The device, apparatus or method of any prior embodiment,
wherein, wherein the labels bound to the amplification surface are
read by counting individual binding events.
[0574] The device, apparatus or method of any prior embodiment,
wherein, wherein the labels bound to the amplification surface are
read by a lump-sum reading method.
[0575] The device, apparatus or method of any prior embodiment,
wherein, wherein the assay has a detection sensitivity of 0.1 nM or
less.
[0576] The device, apparatus or method of any prior embodiment,
wherein, wherein the assay comprises using a sponge to remove
biological materials or labels that are not bound to the
amplification surface.
[0577] The device, apparatus or method of any prior embodiment,
wherein, wherein the signal amplification layer comprises a
D2PA.
[0578] The device, apparatus or method of any prior embodiment,
wherein, wherein the signal amplification layer comprises a layer
of metallic material.
[0579] The device, apparatus or method of any prior embodiment,
wherein, wherein the signal amplification layer comprises a
continuous metallic film that is made of a material selected from
the group consisting of gold, silver, copper, aluminum, alloys
thereof, and combinations thereof.
[0580] The device, apparatus or method of any prior embodiment,
wherein, wherein the different metals layers either locally enhance
or act as a reflector, or both, to enhance an optical signal.
[0581] The device, apparatus or method of any prior embodiment,
wherein, wherein the signal amplification layer comprises a layer
of metallic material and a dielectric material on top of the
metallic material layer, wherein the capture agent is on the
dielectric material.
[0582] The device, apparatus or method of any prior embodiment,
wherein, wherein the metallic material layer is a uniform metallic
layer, nanostructured metallic layer, or a combination.
[0583] The device, apparatus or method of any prior embodiment,
wherein, wherein the amplifies signals by plasmonic
enhancement.
[0584] The device, apparatus or method of any prior embodiment,
wherein, wherein assay comprises detecting the labels by Raman
scattering.
[0585] The device, apparatus or method of any prior embodiment,
wherein, wherein the sample contact area of the first plate further
comprises a site that comprises the proximity-dependent
amplification surface but not the target-specific nucleic acid
probes.
[0586] The device, apparatus or method of any prior embodiment,
wherein the assay comprises calculating a background signal by
reading the site that comprises the proximity-dependent
amplification surface but not the target-specific nucleic acid
probes.
[0587] The device or method of any prior embodiment, wherein the
device further comprise spacers fixed on one of the plate, wherein
the spacers regulate the spacing between the first plate and the
second plate in the closed configuration.
[0588] The device or method of any prior embodiment, wherein the
amplification factor of the surface amplification layer is adjusted
to make the optical signal from a single label that is bound
directly or indirectly to the capture agents visible.
[0589] In the device or method herein described, the spacers can
have a height of 100 nm, 1 um, 5 um, 10 um, 20 um, 50 um, 500 um, 1
mm, or a range between any two of the values; and a preferred range
of 500 nm to 1 um, 1 um to 10 um, or 10 um to 30 um, 30 um to 50
um.
[0590] In the device or method herein described, the spacers can
have a height of 1 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 0.1 s for protein,
0.1 s for nucleic acid (20 bp).
[0591] In the device or method herein described, the spacers can
have a height of 2 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 0.3 s for protein,
0.1 s for nucleic acid (20 bp).
[0592] In the device or method herein described, the spacers can
have a height of 5 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 2 s for protein, 1 s
for nucleic acid (20 bp).
[0593] In the device or method herein described, the spacers can
have a height of 10 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 10 s for protein, 5
s for nucleic acid (20 bp).
[0594] In the device or method herein described, the spacers can
have a height of 30 um or less, thereby the saturation incubation
time (or saturation binding time) is less than for 60 s protein, 30
s for nucleic acid (20 bp).
[0595] In the device or method herein described, the spacers can
have a height of 50 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 2 min for protein, 1
min for nucleic acid (20 bp).
[0596] In the device or method herein described, the spacers can
have a height of 100 um or less, thereby the saturation incubation
time (or saturation binding time) is less than 10 min for protein,
5 min for nucleic acid (20 bp).
Related Documents
[0597] The present invention includes a variety of embodiments,
which can be combined in multiple ways as long as the various
components do not contradict one another. The embodiments should be
regarded as a single invention file: each filing has other filing
as the references and is also referenced in its entirety and for
all purpose, rather than as a discrete independent. These
embodiments include not only the disclosures in the current file,
but also the documents that are herein referenced, incorporated, or
to which priority is claimed.
(10) Definitions
[0598] The terms used in describing the devices, systems, and
methods herein disclosed are defined in the current application, or
in PCT Application (designating U.S.) Nos. PCT/US2016/045437 and
PCT/US0216/051775, which were respectively filed on Aug. 10, 2016
and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065,
which was filed on Feb. 7, 2017, U.S. Provisional Application No.
62/456,287, which was filed on Feb. 8, 2017, and U.S. Provisional
Application No. 62/456,504, which was filed on Feb. 8, 2017, all of
which applications are incorporated herein in their entireties for
all purposes.
[0599] The terms "CROF Card (or card)", "COF Card", "QMAX-Card",
"Q-Card", "CROF device", "COF device", "QMAX-device", "CROF
plates", "COF plates", and "QMAX-plates" are interchangeable,
except that in some embodiments, the COF card does not comprise
spacers; and the terms refer to a device that comprises a first
plate and a second plate that are movable relative to each other
into different configurations (including an open configuration and
a closed configuration), and that comprises spacers (except some
embodiments of the COF card) that regulate the spacing between the
plates. The term "X-plate" refers to one of the two plates in a
CROF card, wherein the spacers are fixed to this plate. More
descriptions of the COF Card, CROF Card, and X-plate are given in
the provisional application Ser. No. 62/456,065, filed on Feb. 7,
2017, which is incorporated herein in its entirety for all
purposes.
(11) Q-Card, Spacer and Uniform Sample Thickness
[0600] The devices, systems, and methods herein disclosed can
include or use Q-cards, spacers, and uniform sample thickness
embodiments for sample detection, analysis, and quantification. In
some embodiments, the Q-card comprises spacers, which help to
render at least part of the sample into a layer of high uniformity.
The structure, material, function, variation and dimension of the
spacers, as well as the uniformity of the spacers and the sample
layer, are herein disclosed, or listed, described, and summarized
in PCT Application (designating U.S.) Nos. PCT/US2016/045437 and
PCT/US0216/051775, which were respectively filed on Aug. 10, 2016
and Sep. 14, 2016, U.S. Provisional Application No. 62/456,065,
which was filed on Feb. 7, 2017, U.S. Provisional Application No.
62/456,287, which was filed on Feb. 8, 2017, and U.S. Provisional
Application No. 62/456,504, which was filed on Feb. 8, 2017, all of
which applications are incorporated herein in their entireties for
all purposes.
(12) Hinges, Opening Notches, Recessed Edge and Sliders
[0601] The devices, systems, and methods herein disclosed can
include or use Q-cards for sample detection, analysis, and
quantification. In some embodiments, the Q-card comprises hinges,
notches, recesses, and sliders, which help to facilitate the
manipulation of the Q card and the measurement of the samples. The
structure, material, function, variation and dimension of the
hinges, notches, recesses, and sliders are herein disclosed, or
listed, described, and summarized in PCT Application (designating
U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/456,287, which was filed
on Feb. 8, 2017, and U.S. Provisional Application No. 62/456,504,
which was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
(13) Q-Card, Sliders, and Smartphone Detection System
[0602] The devices, systems, and methods herein disclosed can
include or use Q-cards for sample detection, analysis, and
quantification. In some embodiments, the Q-cards are used together
with sliders that allow the card to be read by a smartphone
detection system. The structure, material, function, variation,
dimension and connection of the Q-card, the sliders, and the
smartphone detection system are herein disclosed, or listed,
described, and summarized in PCT Application (designating U.S.)
Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/456,287, which was filed
on Feb. 8, 2017, and U.S. Provisional Application No. 62/456,504,
which was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
(14) Detection Methods
[0603] The devices, systems, and methods herein disclosed can
include or be used in various types of detection methods. The
detection methods are herein disclosed, or listed, described, and
summarized in PCT Application (designating U.S.) Nos.
PCT/US2016/045437 and PCT/US0216/051775, which were respectively
filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional
Application No. 62/456,065, which was filed on Feb. 7, 2017, U.S.
Provisional Application No. 62/456,287, which was filed on Feb. 8,
2017, and U.S. Provisional Application No. 62/456,504, which was
filed on Feb. 8, 2017, all of which applications are incorporated
herein in their entireties for all purposes.
(15) Labels, Capture Agent and Detection Agent
[0604] The devices, systems, and methods herein disclosed can
employ various types of labels, capture agents, and detection
agents that are used for analytes detection. The labels are herein
disclosed, or listed, described, and summarized in PCT Application
(designating U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775,
which were respectively filed on Aug. 10, 2016 and Sep. 14, 2016,
U.S. Provisional Application No. 62/456,065, which was filed on
Feb. 7, 2017, U.S. Provisional Application No. 62/456,287, which
was filed on Feb. 8, 2017, and U.S. Provisional Application No.
62/456,504, which was filed on Feb. 8, 2017, all of which
applications are incorporated herein in their entireties for all
purposes.
(16) Analytes
[0605] The devices, systems, and methods herein disclosed can be
applied to manipulation and detection of various types of analytes
(including biomarkers). The analytes and are herein disclosed, or
listed, described, and summarized in PCT Application (designating
U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/456,287, which was filed
on Feb. 8, 2017, and U.S. Provisional Application No. 62/456,504,
which was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
(17) Applications (Field and Samples)
[0606] The devices, systems, and methods herein disclosed can be
used for various applications (fields and samples). The
applications are herein disclosed, or listed, described, and
summarized in PCT Application (designating U.S.) Nos.
PCT/US2016/045437 and PCT/US0216/051775, which were respectively
filed on Aug. 10, 2016 and Sep. 14, 2016, U.S. Provisional
Application No. 62/456,065, which was filed on Feb. 7, 2017, U.S.
Provisional Application No. 62/456,287, which was filed on Feb. 8,
2017, and U.S. Provisional Application No. 62/456,504, which was
filed on Feb. 8, 2017, all of which applications are incorporated
herein in their entireties for all purposes.
(18) Cloud
[0607] The devices, systems, and methods herein disclosed can
employ cloud technology for data transfer, storage, and/or
analysis. The related cloud technologies are herein disclosed, or
listed, described, and summarized in PCT Application (designating
U.S.) Nos. PCT/US2016/045437 and PCT/US0216/051775, which were
respectively filed on Aug. 10, 2016 and Sep. 14, 2016, U.S.
Provisional Application No. 62/456,065, which was filed on Feb. 7,
2017, U.S. Provisional Application No. 62/456,287, which was filed
on Feb. 8, 2017, and U.S. Provisional Application No. 62/456,504,
which was filed on Feb. 8, 2017, all of which applications are
incorporated herein in their entireties for all purposes.
[0608] Without any intention to limit the use of the present method
and device, in some embodiments, the method may be employed to
identify a microbial pathogen from a clinical sample. In these
embodiments, the target sequences may be from multiple different
pathogens (e.g., at least 10 or at least 100 different pathogens),
without knowing which pathogen is responsible for an infection,
Microbes that might be identified using the present methods,
compositions and kits include but are not limited to: a plurality
of species of Gram (+) bacteria, plurality of species of Gram (-)
bacteria, a plurality of species of bacteria in the family
Enterobacteriaceae, a plurality of species of bacteria in the genus
Enterococcus, a plurality of species of bacteria in the genus
Staphylococcus, and a plurality of species of bacteria in the genus
Campylobacter, Escherichia coli (E. coli), E. coli of various
strains such as, K12-MG1655, CFT073, O157:H7 EDL933, O157:H7
VT2-Sakai, etc., Streptococcus pneumoniae, Pseudomonas aeruginosa,
Staphylococcus aureus, coagulase-negative staphylococci, a
plurality of Candida species including C. albicans, C. tropicalis,
C. dubliniensis, C. viswanathii, C. parapsilosis, Klebsiella
pneumoniae, a plurality of Mycobacterium species such as M.
tuberculosis, M. bovis, M. bovis BCG, M. scrofulaceum, M. kansasii,
M. chelonae, M. gordonae, M. ulcerans, M. genavense, M. xenoi, M.
simiae, M. fortuitum, M. malmoense, M. celatum, M. haemophilum and
M. africanum, Listeria species, Chlamydia species, Mycoplasma
species, Salmonella species, Brucella species, Yersinia species,
etc. Thus, the subject method enables identification of microbes to
the level of the genus, species, sub-species, strain or variant of
the microbe.
Additional Notes
[0609] Further examples of inventive subject matter according to
the present disclosure are described in the following enumerated
paragraphs.
[0610] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
referents unless the context clearly dictates otherwise, e.g., when
the word "single" is used. For example, reference to "an analyte"
includes a single analyte and multiple analytes, reference to "a
capture agent" includes a single capture agent and multiple capture
agents, reference to "a detection agent" includes a single
detection agent and multiple detection agents, and reference to "an
agent" includes a single agent and multiple agents.
[0611] As used herein, the terms "adapted" and "configured" mean
that the element, component, or other subject matter is designed
and/or intended to perform a given function. Thus, the use of the
terms "adapted" and "configured" should not be construed to mean
that a given element, component, or other subject matter is simply
"capable of" performing a given function. Similarly, subject matter
that is recited as being configured to perform a particular
function may additionally or alternatively be described as being
operative to perform that function.
[0612] As used herein, the phrase, "for example," the phrase, "as
an example," and/or simply the terms "example" and "exemplary" when
used with reference to one or more components, features, details,
structures, embodiments, and/or methods according to the present
disclosure, are intended to convey that the described component,
feature, detail, structure, embodiment, and/or method is an
illustrative, non-exclusive example of components, features,
details, structures, embodiments, and/or methods according to the
present disclosure. Thus, the described component, feature, detail,
structure, embodiment, and/or method is not intended to be
limiting, required, or exclusive/exhaustive; and other components,
features, details, structures, embodiments, and/or methods,
including structurally and/or functionally similar and/or
equivalent components, features, details, structures, embodiments,
and/or methods, are also within the scope of the present
disclosure.
[0613] As used herein, the phrases "at least one of" and "one or
more of," in reference to a list of more than one entity, means any
one or more of the entity in the list of entity, and is not limited
to at least one of each and every entity specifically listed within
the list of entity. For example, "at least one of A and B" (or,
equivalently, "at least one of A or B," or, equivalently, "at least
one of A and/or B") may refer to A alone, B alone, or the
combination of A and B.
[0614] As used herein, the term "and/or" placed between a first
entity and a second entity means one of (1) the first entity, (2)
the second entity, and (3) the first entity and the second entity.
Multiple entity listed with "and/or" should be construed in the
same manner, i.e., "one or more" of the entity so conjoined. Other
entity may optionally be present other than the entity specifically
identified by the "and/or" clause, whether related or unrelated to
those entities specifically identified.
[0615] Where numerical ranges are mentioned herein, the invention
includes embodiments in which the endpoints are included,
embodiments in which both endpoints are excluded, and embodiments
in which one endpoint is included and the other is excluded. It
should be assumed that both endpoints are included unless indicated
otherwise. Furthermore, unless otherwise indicated or otherwise
evident from the context and understanding of one of ordinary skill
in the art.
[0616] In the event that any patents, patent applications, or other
references are incorporated by reference herein and (1) define a
term in a manner that is inconsistent with and/or (2) are otherwise
inconsistent with, either the non-incorporated portion of the
present disclosure or any of the other incorporated references, the
non-incorporated portion of the present disclosure shall control,
and the term or incorporated disclosure therein shall only control
with respect to the reference in which the term is defined and/or
the incorporated disclosure was present originally.
Aspects
[0617] Aspect 1. A method for performing a homogeneous nucleic acid
detection assay comprising: [0618] (a) obtaining a QMAX device
comprising a first plate and a second plate, wherein the first
plate and the second plate, each comprises a sample contacting area
for contacting a sample that contains one or more target nucleic
acids; [0619] the first plate comprises, on its sample contacting
area, a binding site that comprises: [0620] (i) surface
amplification surface; and [0621] (ii) target-specific nucleic acid
probes that are immobilized on said amplification surface and that
specifically binds to a part of the target nucleic acid; and the
second plate comprises a sample contact area comprising a reagent
storage site that comprises target-specific nucleic acid detection
agents that specifically binds to another part of the target
nucleic acid; [0622] (b) depositing the sample on one or both of
the plates when the plates are in an open configuration; [0623] (c)
closing the plates to a closed configuration; and. [0624] (d),
after (c), while the plates remain in the closed configuration and
without any washing step, detecting the target nucleic acid by
reading the sample contact area with a reading device to produce an
image of signals; [0625] wherein: (i) the thickness of the sample
in the closed configuration, (ii) the concentration of labels
dissolved in the sample in the closed configuration, and (iii) the
amplification factor of the proximity-dependent amplification
surface are configured such that labels that are indirectly bound
to the nucleic acid probes via a target nucleic acid are visible
without washing away any biological materials or labels that are
not bound to the surface amplification surface; [0626] wherein one
of the configurations is an open configuration, in which the
average spacing between the inner surfaces of the two plates is at
least 200 um; and [0627] wherein another of the configurations is a
close configuration, in which, at least part of the sample is
between the two plates and the average spacing between the inner
surfaces of the plates is less than 200 um. [0628] Aspect 2. A
device for analyzing a homogenous sample comprising: [0629] a first
plate, a second plate, and a binding site, wherein [0630] (a) the
first and second plates are movable relative to each other into
different configurations, and have, on its respective surface, a
sample contact area for contacting a sample that contains a target
analyte, [0631] (b) the sample contact area on the first plate has
a binding site that comprises: [0632] (i) proximity-dependent
signal amplification layer, and [0633] (ii) target-specific nucleic
acid probes that are attached to said proximity-dependent signal
amplification layer that bind to part of a target nucleic acid;
[0634] (c) the sample contact area on the second plate comprising a
reagent storage site that comprises target-specific nucleic acid
detection agents that bind to another part of the target nucleic
acid; [0635] wherein one of the configurations is an open
configuration; [0636] wherein another of the configurations is a
close configuration, in which, at least part of the sample is
between the two plates; and [0637] wherein the thickness of the
sample in the closed configuration, the concentration of the labels
dissolved in the sample in the closed configuration, and the
amplification factor of the proximity-dependent signal
amplification layer are configured such that any the labels that
are indirectly bound to the target-specific nucleic acid probes are
visible without washing away of the unbound labels. [0638] Aspect
3. An apparatus comprising a thermal cycler and a device of Aspect
2. [0639] Aspect 4. An apparatus comprising a thermal cycler, a
device of Aspect 2, and a reader for real-time PCR. [0640] Aspect
5. A method for rapid nucleic acid detection assay comprising:
[0641] (a) obtaining a QMAX device comprising a first plate and a
second plate, wherein the first plate and the second plate, each
comprises a sample contacting area for contacting a sample that
contains one or more target nucleic acids; [0642] the first plate
comprises, on its sample contacting area, a binding site that
comprises target-specific nucleic acid probes that are immobilized
on the site and that specifically binds to part of the target
nucleic acid; and [0643] the second plate comprises a sample
contact area comprising a reagent storage site that comprises
target-specific nucleic acid detection agents that specifically
binds to another part of the target nucleic acid; [0644] (b)
depositing the sample on one or both of the plates when the plates
are in an open configuration; [0645] (c) closing the plates to a
closed configuration for incubation for a period of time; and.
[0646] (d) opening the plates and pressing the plate again a
washing sponge that has washing solution for a period of time and
then releasing the washing sponge; [0647] (e), after (d), reading
the sample contact area with a reading device to produce an image
of signals; [0648] wherein: (i) the thickness of the sample in the
closed configuration, (ii) the concentration of labels dissolved in
the sample in the closed configuration, and (iii) the amplification
factor of the proximity-dependent amplification surface are
configured such that labels that are indirectly bound to the
nucleic acid probes via a target nucleic acid are visible without
washing away any biological materials or labels that are not bound
to the proximity-dependent amplification surface; wherein one of
the configurations is an open configuration, in which the average
spacing between the inner surfaces of the two plates is at least
200 um; and [0649] wherein another of the configurations is a close
configuration, in which, at least part of the sample is between the
two plates and the average spacing between the inner surfaces of
the plates is less than 200 um. [0650] Aspect 6. The device,
apparatus or method of any prior Aspects, wherein the spacing
between the first plate and the second plate in the closed
configuration is configured to make saturation binding time of the
target analyte to the capture agents 300 sec or less. [0651] Aspect
7. The device, apparatus or method of any prior Aspects, wherein
the spacing between the first plate and the second plate in the
closed configuration is configured to make saturation binding time
of the target analyte to the capture agents 300 sec or less. [0652]
Aspect 8. The device, apparatus or method of any prior Aspects,
wherein the spacing between the first plate and the second plate in
the closed configuration is configured to make saturation binding
time of the target analyte to the capture agents 60 sec or less.
[0653] Aspect 9. The device, apparatus or method of any prior
Aspects, wherein the target nucleic acid is a DNA or RNA, including
genomic DNA, cfDNA, cDNA ctDNA, mRNA and miRNA. [0654] Aspect 10.
The device, apparatus or method of any prior Aspects, wherein the
time from step (b) to obtaining a result is less than 10 min.
[0655] Aspect 11. The device, apparatus or method of any prior
Aspects, wherein the thickness of the sample in the closed
configuration, the concentration of the labels dissolved in the
sample in the closed configuration, and the amplification factor of
the surface amplification layer are configured such that any the
labels that are bound directly or indirectly to the probles are
visible in the closed configuration without washing away of the
unbound labels. [0656] Aspect 12. The device, apparatus or method
of any prior Aspects, wherein he labels bound to the
proximity-dependent amplification surface are visible in less than
60 seconds. [0657] Aspect 13. The device, apparatus or method of
any prior Aspects, wherein, wherein the labels bound to the
proximity-dependent amplification surface are visible in less than
60 seconds. [0658] Aspect 14. The device, apparatus or method of
any prior Aspects, wherein, wherein the storage site is
approximately above the binding site on the first plate in the
closed configuration. [0659] Aspect 15. The device, apparatus or
method of any prior Aspects, wherein, wherein the target-specific
nucleic acid probes and the target-specific nucleic acid detection
agents form a sandwich that comprises the label. [0660] Aspect 16.
The device, apparatus or method of any prior Aspects, wherein,
wherein the signals are read without using a wash step to remove
any biological materials or labels that are not bound to the
amplification surface. [0661] Aspect 17. The device, apparatus or
method of any prior Aspects, wherein, wherein the labels bound to
the amplification surface are read by counting individual binding
events. [0662] Aspect 18. The device, apparatus or method of any
prior Aspects, wherein, wherein the labels bound to the
amplification surface are read by a lump-sum reading method. [0663]
Aspect 19. The device, apparatus or method of any prior Aspects,
wherein, wherein the assay has a detection sensitivity of 0.1 nM or
less. [0664] Aspect 20. The device, apparatus or method of any
prior Aspects, wherein, wherein the assay comprises using a sponge
to remove biological materials or labels that are not bound to the
amplification surface. [0665] Aspect 21. The device, apparatus or
method of any prior Aspects, wherein, wherein the signal
amplification layer comprises a D2PA. [0666] Aspect 22. The device,
apparatus or method of any prior Aspects, wherein, wherein the
signal amplification layer comprises a layer of metallic material.
[0667] Aspect 23. The device, apparatus or method of any prior
Aspects, wherein, wherein the signal amplification layer comprises
a continuous metallic film that is made of a material selected from
the group consisting of gold, silver, copper, aluminum, alloys
thereof, and combinations thereof. [0668] Aspect 24. The device,
apparatus or method of any prior Aspects, wherein, wherein the
different metals layers either locally enhance or act as a
reflector, or both, to enhance an optical signal. [0669] Aspect 25.
The device, apparatus or method of any prior Aspects, wherein,
wherein the signal amplification layer comprises a layer of
metallic material and a dielectric material on top of the metallic
material layer, wherein the capture agent is on the dielectric
material. [0670] Aspect 26. The device, apparatus or method of any
prior Aspects, wherein, wherein the metallic material layer is a
uniform metallic layer, nanostructured metallic layer, or a
combination. [0671] Aspect 27. The device, apparatus or method of
any prior Aspects, wherein, wherein the amplifies signals by
plasmonic enhancement. [0672] Aspect 28. The device, apparatus or
method of any prior Aspects, wherein, wherein assay comprises
detecting the labels by Raman scattering. [0673] Aspect 29. The
device, apparatus or method of any prior Aspects, wherein, wherein
the sample contact area of the first plate further comprises a site
that comprises the proximity-dependent amplification surface but
not the target-specific nucleic acid probes. [0674] Aspect 30. The
device, apparatus or method of any prior Aspects, wherein the assay
comprises calculating a background signal by reading the site that
comprises the proximity-dependent amplification surface but not the
target-specific nucleic acid probes. [0675] Aspect 31. The device
or method of any prior Aspects, wherein the device further comprise
spacers fixed on one of the plates, wherein the spacers regulate
the spacing between the first plate and the second plate in the
closed configuration. [0676] Aspect 32. The device or method of any
prior Aspects, wherein the amplification factor of the surface
amplification layer is adjusted to make the optical signal from a
single label that is bound directly or indirectly to the capture
agents visible.
* * * * *