U.S. patent application number 15/031062 was filed with the patent office on 2016-09-08 for enrichment and detection of nucleic acids with ultra-high sensitivity.
The applicant listed for this patent is COMPLIANCE DECISIONS, INC., THE REGENTS OF THE UNIVERSITY OF CALIFORNIA. Invention is credited to Ian Lian, Yu-Hwa Lo, Wen Qiao, Tony Minghung Yen, Tiantian Zhang.
Application Number | 20160258020 15/031062 |
Document ID | / |
Family ID | 52993469 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160258020 |
Kind Code |
A1 |
Lo; Yu-Hwa ; et al. |
September 8, 2016 |
ENRICHMENT AND DETECTION OF NUCLEIC ACIDS WITH ULTRA-HIGH
SENSITIVITY
Abstract
Methods, systems, and devices are disclosed for enrichment and
detection of molecules of a target biomarker. In one aspect, In one
aspect, a biosensor device for enriching and detecting biomarker
molecules include a substrate, and a microarray of hydrophilic
islands disposed on the substrate. A sensing area on each of the
microarray hydrophilic islands is structured to anchor
bio-molecular probes of at least one type for detecting molecules
of a target biomarker and to attract an array of nanodroplets of a
biomarker solution that includes the target biomarker molecules. A
hydrophobic surface disposed to surround the microarray of
hydrophilic islands.
Inventors: |
Lo; Yu-Hwa; (San Diego,
CA) ; Qiao; Wen; (Suzhou, CN) ; Zhang;
Tiantian; (San Diego, CA) ; Lian; Ian;
(Alhambra, CA) ; Yen; Tony Minghung; (Santa Clara,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF CALIFORNIA
COMPLIANCE DECISIONS, INC. |
Oakland
Irvine |
CA
CA |
US
US |
|
|
Family ID: |
52993469 |
Appl. No.: |
15/031062 |
Filed: |
October 21, 2014 |
PCT Filed: |
October 21, 2014 |
PCT NO: |
PCT/US2014/061639 |
371 Date: |
April 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61893532 |
Oct 21, 2013 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6825 20130101;
B01L 2300/0819 20130101; C12Q 1/6883 20130101; B01L 2300/0887
20130101; C12Q 2600/178 20130101; C12Q 1/6837 20130101; C12Q 1/6837
20130101; B01L 3/5085 20130101; B01L 2300/161 20130101; B01L
2300/166 20130101; B01L 3/5088 20130101; B01L 2300/0636 20130101;
C12Q 1/6825 20130101; C12Q 2565/501 20130101; C12Q 2565/607
20130101; C12Q 2563/159 20130101; C12Q 2565/501 20130101; C12Q
2563/159 20130101; C12Q 2565/607 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; B01L 3/00 20060101 B01L003/00 |
Claims
1. A biosensor device for enriching and detecting biomarker
molecules, the biosensor device comprising: a substrate; a
microarray of hydrophilic islands disposed on the substrate;
sensing areas disposed on the microarray hydrophilic islands, the
sensing areas structured to anchor bio-molecular probes of at least
one type for detecting molecules of a target biomarker and to
attract an array of droplets of a biomarker solution that includes
the target biomarker molecules; and a hydrophobic surface
surrounding the microarray of hydrophilic islands, the hydrophobic
surface including nanostructures having rough surfaces to enrich
the target biomarker molecules on the sensing area by enhancing
evaporation of the array of droplets leading to an enriched array
of droplets with an increased concentration of the target biomarker
molecules compared to before evaporation; wherein the sensing areas
are structured to receive a layer of water-immiscible-liquid over
the enriched array of droplets to form water-immiscible-liquid
encapsulated reaction chambers for controlling a reaction between
the target biomarker molecules and the bio-molecular probes.
2. The biosensor device of claim 1, wherein the sensing areas
include a layer of a hydrophilic material.
3. The biosensor device of claim 2, wherein the hydrophilic
material of the sensing areas includes a dielectric material.
4. The biosensor device of claim 2, wherein the hydrophilic
material of the sensing areas includes silicon oxide
(SiO.sub.2).
5. The biosensor device of claim 1, wherein the nanostructures of
the hydrophobic surface includes nanopillars.
6. The biosensor device of claim 1, wherein the sensing areas
structured to receive a labeling material to label target biomarker
molecules that reacted with the bio-molecular probes.
7. The biosensor device of claim 3, wherein the labeling material
includes quantum dots.
8. The biosensor device of claim 1, wherein the hydrophilic
property of the sensing areas causes the array of droplets to be
self-aligned with the sensing areas.
9. The biosensor device of claim 1, wherein the sensing areas
covered microarray of hydrophilic islands and the nanostructures of
the hydrophobic surface are disposed to have a height difference
that reduces adhesion of target biomolecules to a sidewall of the
sensing areas covered microarray of hydrophilic islands during
evaporation.
10. The biosensor device of claim 1, wherein the hydrophobic
surface includes black silicon.
11. The biosensor device of claim 1, wherein the bio-molecular
probes of at least one kind include DNA probes and the target
biomarker molecules include target nucleic acids.
12. The biosensor device of claim 1, wherein the sensing areas of
the microarray of hydrophilic islands are structured to enrich and
detect different types of target biomarker molecules.
13. The biosensor device of claim 1, wherein the biosensor device
is configured to detect the target biomarker molecules of
approximately 1 femtomolar (fM) concentration.
14. The biosensor device of claim 1, wherein the biosensor device
is configured to detect the target biomarker molecules of
approximately 0.5 fM concentration.
15. The biosensor device of claim 1, wherein the biosensor device
is configured to enrich and detect multiple biomarker molecule
types in parallel.
16. The biosensor device of claim 11, wherein the multiple types of
biomarker molecules includes RNA and DNA markers.
17. The biosensor device of claim 1, wherein each droplet is
nanoliter or less.
18. The biosensor device of claim 1, wherein the
water-immiscible-liquid includes oil.
19. A method performed by a biosensor device to enrich and detect
biomarker molecules, the method comprising: receiving, by a
biosensor device including a microarray of hydrophilic islands
having sensing areas and surrounded by hydrophobic nanostructures,
bio-molecular probes of at least one type for detecting molecules
of a target biomarker to functionalize the sensing areas of the
microarray of hydrophilic islands; receiving, over the
functionalized sensing areas of the microarray of hydrophilic
islands, droplets of a biomarker solution that includes the
biomarker molecules to form an array of droplets of the biomarker
solutions on the functionalized sensing areas; receiving over the
array of droplets of the biomarker solutions a layer of
water-immiscible-liquid to encapsulate the array of droplets of the
biomarker solutions to form water-immiscible-liquid encapsulated
reaction chambers for controlling a reaction between the target
biomarker molecules and the bio-molecular probes.
20. The method of claim 19, wherein the bio-molecular probes of at
least one type include DNA probes, and the molecules of biomarkers
include nucleic acids.
21. The method of claim 20, wherein the DNA probes include DNA
oligonucleotides.
22. The method of claim 20, wherein receiving the layer of
water-immiscible-liquid to encapsulate the array of droplets of the
biomarker solutions to form water-immiscible-liquid encapsulated
reaction chambers for controlling a reaction between the target
biomarker molecules and the bio-molecular probes include
facilitating hybridization of the biomarker molecules with the DNA
probes within the nano-chambers.
23. The method of claim 19, comprising: receiving labeling
materials to within the reaction chambers to label bio-molecular
probe attached biomarker molecules formed responsive to the
controlled reaction.
24. The method of claim 23, wherein the labeling materials include
quantum-dots.
25. The method of claim 20, wherein the nucleic acids include DNA,
RNA or miRNA-based nucleic acids.
26. The method of claim 19, wherein the sensing areas include a
layer of silicon oxide (SiO.sub.2).
27. The method of claim 19, wherein the hydrophobic surface
includes black silicon.
28. The method of claim 19, wherein the target biomarker molecules
includes fluorescently labeled biomarker molecules to determine a
concentration of the target biomarker molecules based on an
fluorescent intensity of the fluorescently labeled biomarker
molecules that react with bio-molecular probes.
29. The method of claim 19, wherein each droplet is nanoliter or
less.
30. The method of claim 19, wherein the water-immiscible-liquid
includes oil.
31. A method performed by a biosensor device for enriching
biomarker molecules to facilitate ultra-sensitive detection and
quantification of the biomarker molecules, the method comprising:
receiving, by a biosensor device including an microarray of
hydrophilic islands having sensing areas and surrounded by a
hydrophobic structure, bio-molecular probes of at least one type
for detecting molecules of a target biomarker to functionalize the
sensing areas of the microarray of hydrophilic islands; receiving,
over the functionalized sensing areas of the microarray of
hydrophilic islands, droplets of a biomarker solution that includes
the biomarker molecules to form droplets of the biomarker molecules
that cover the sensing areas of the microarray of hydrophilic
islands; allowing the biomarker solution to evaporate over the
microarray of hydrophilic islands to concentrate the biomarker
molecules onto the sensing areas of the microarray of hydrophilic
islands; receiving, over the functionalized sensing areas of the
microarray of hydrophilic islands containing the concentrated
biomarker molecules, a liquid in a controlled time to form an array
of self-assembled droplets to resuspend the concentrated biomarker
molecules in the droplets over the functionalized sensing areas;
receiving, over the array of self-assembled droplets of the
biomarker solutions, a layer of water-immiscible-liquid to
encapsulate the array of self-assembled droplets to form
water-immiscible-liquid encapsulated reaction chambers for
controlling a reaction between the target biomarker molecules and
the bio-molecular probes; and allowing a reaction between the
target biomarker molecules and the bio-molecular probes to form
hybridized target biomarker molecules.
32. The method of claim 31, wherein the liquid includes one of: a
hybridization buffer containing a labeling material for labeling
the target biomarker molecules that have reacted with the
bio-molecular probes; or water.
33. The method claim 32, wherein the labeling material includes
quantum-dots linked reporting DNAs.
34. The method claim 31, wherein the microarray of hydrophilic
islands are separated by a grid of hydrophobic nanostructures.
35. The method claim 34, wherein nanostructures include
nanopillars.
36. The method of claim 34, wherein the grid of hydrophobic
nanostructures include Teflon-coated grids.
37. The method claim 31, wherein the biomarker solution includes a
hybridization buffer of the biomarker molecules.
38. The method of claim 31, wherein the biomarker containing
solution includes miRNA containing RNAse-free deionized water.
39. The method of claim 31, wherein the target biomarker includes
DNAs, RNAs, miRNAs, synthetic miR-205 DNA mimic, or other nucleic
acids.
40. The method of claim 31, wherein the biosensor device includes a
lab-on-a-chip device.
41. The method of claim 31, wherein the hydrophobic structure
includes a nanopillar structure made of black silicon.
42. The method of claim 31, wherein the sensing areas of the
microarray of hydrophilic islands includes a layer of
SiO.sub.2.
43. The method of claim 31, wherein each of the microarray of
hydrophilic islands is configured to detect and quantify a
different typical of target biomarker.
44. The method of claim 31, wherein the biosensor device is
configured to perform parallel operation with a large number of
target biomarkers.
45. The method of claim 31, wherein the biosensor device is
configured to detect a target biomarker with a detection
sensitivity of approximately 0.05 femtomolar (fM).
46. The method of claim 31, wherein the controlled time is
approximately 1 second.
47. The method of claim 31, wherein after the completion of the
hybridization, the method further includes receiving, at the
functionalized sensing areas, a cleaning liquid to remove the layer
of oil and liquid to expose the hybridized target biomarker and the
bio-molecular probes.
48. The method of claim 31, wherein allowing the biomarker solution
to evaporate over the microarray of hydrophilic islands to
concentrate the biomarker molecules includes allowing the liquid in
the biomarker solution to completely dry.
49. The method of claim 31, wherein allowing the biomarker solution
to evaporate over the microarray of hydrophilic islands to
concentrate the biomarker molecules includes allowing the biomarker
solution to concentrate to a predetermined thickness.
50. The method of claim 48, wherein the predetermined thickness is
approximately 20 .mu.m.
51. The method of claim 31, wherein each droplet is nanoliter or
less.
52. The method of claim 31, wherein the water-immiscible-liquid
includes oil.
Description
PRIORITY CLAIM AND RELATED PATENT APPLICATION
[0001] This patent document claims priority to and the benefit of
U.S. Provisional Application No. 61/893,532 entitled "METHODS AND
DEVICES FOR ENRICHMENT AND DETECTION OF NUCLEIC ACIDS" and filed
Oct. 21, 2013, the entire contents of which are incorporated by
reference in this document.
TECHNICAL FIELD
[0002] This patent document relates to biosensor technologies and
analytical devices.
BACKGROUND
[0003] A biological sensor or biosensor is an analytical tool that
can detect a chemical, substance, or organism using a biologically
sensitive component coupled with a transducing element to convert a
detection event into a signal for processing and/or display.
Biosensors can use biological materials as the biologically
sensitive component, e.g., such as biomolecules including enzymes,
antibodies, aptamers, peptides, nucleic acids, etc., or small
molecules such as carbohydrates, as well as virus and living cells.
For example, molecular biosensors can use specific chemical
properties or molecular recognition mechanisms to identify target
agents. Biosensors can use the transducer element to transform a
signal resulting from the detection of an analyte by the
biologically sensitive component into a different signal that can
be addressed by a suitable transduction mechanism, for example,
electrical, magnetic, mechanical, physicochemical, electrochemical,
optical, piezoelectric, or others.
[0004] Detection of low abundance biomolecules is challenging for
biosensors that rely on surface chemical reactions. For surface
reaction based biosensors, it often takes hours or even days for
biomolecules of diffusivities in the order of 10.sup.-10-11
m.sup.2/sec to reach the surface of the sensors through Brownian
motion. Moreover, the repulsive Coulomb interactions between the
molecules and the probes can further defer the binding process,
leading to undesirably long detection time for applications such as
point-of-care in-vitro diagnosis.
SUMMARY
[0005] Techniques, systems, and devices are disclosed for
enrichment and detection of biomarker molecules including nucleic
acids.
[0006] The subject matter described in this patent document can be
implemented in specific ways that provide one or more of the
following features. For example, the disclosed devices and systems
are capable of enriching and detection of biomarkers molecules,
e.g., such as DNAs and RNAs, without requiring any amplification
steps to increase the copy number of those molecular markers. Such
devices are especially attractive to a variety of applications, for
example, including, but not limited to, gene expression analysis by
estimating the copy numbers of cDNA or mRNA transcripts present in
the sample; applications in molecular detection, nucleic acid
biomarker quantification for clinical, laboratory and point-of-care
diagnostic purposes; development to detect pathogen and infectious
disease with direct identification of endogenous sequences,
including fragmental microbial genome (DNA) and retroviral RNA
sequences; capability to quantify miRNA and other forms of
circulating epigenetic marker for early diagnosis of disease and
injury; and mutational analysis of cancer and hereditary diseases
using tiling probes layout.
[0007] In one aspect, a biosensor device for enriching and
detecting biomarker molecules include a substrate, and a microarray
of hydrophilic islands disposed on the substrate. A layer of
silicon oxide (SiO2) is disposed over the microarray of hydrophilic
islands to form a sensing area on the microarray hydrophilic
islands, the sensing areas are structured to anchor bio-molecular
probes of at least one type for detecting molecules of a target
biomarker and to attract an array of droplets of a biomarker
solution that includes the target biomarker molecules. A
super-hydrophobic surface is disposed to surround the microarray of
hydrophilic islands. The super-hydrophobic surface include
nanostructures having rough surfaces to enrich the target biomarker
molecules on the sensing area by enhancing evaporation of the array
of droplets leading to an enriched array of droplets with an
increased concentration of the target biomarker molecules compared
to before evaporation. The sensing area is structured to receive a
layer of water-immiscible-liquid over the enriched array of
droplets to form water-immiscible-liquid encapsulated reaction
chambers (e.g., nano or microliter volume) for controlling a
reaction between the target biomarker molecules and the
bio-molecular probes. In one embodiment, the
water-immiscible-liquid includes oil. In one embodiment, each
droplet is nanoliter or less.
[0008] The biosensor can be implemented in various ways to include
one or more of the following features. The nanostructures of the
super-hydrophobic surface can include nanopillars. The sensing area
can be structured to receive a labeling material to label target
biomarker molecules that reacted with the bio-molecular probes. The
labeling material can include quantum dots. The hydrophilic
property of the SiO2 layer can cause the array of droplets to be
self-aligned with the sensing area. The SiO2 layer covered
microarray of hydrophilic islands and the nanostructures of the
super-hydrophobic surface can be disposed to have a height
difference that reduces adhesion of target biomolecules to a
sidewall of the SiO2 layer covered microarray of hydrophilic
islands during evaporation. The super-hydrophobic surface can
include black silicon. The bio-molecular probes of at least one
kind can include DNA probes and the target biomarker molecules can
include target nucleic acids. The sensing areas of the microarray
of hydrophilic islands can be structured to enrich and detect
different types of target biomarker molecules. The biosensor device
can detect the biomarker molecules of approximately 0.5 femtomolar
(fM) concentration. The biosensor device can enrich and detect
multiple biomarker molecule types in parallel. The multiple types
of biomarker molecules can include RNA and DNA markers. In one
embodiment, each droplet is nanoliter or less
[0009] In another aspect, a method performed by a biosensor device
to enrich and detect biomarker molecules includes receiving, by a
biosensor device including a microarray of hydrophilic islands
having sensing surfaces and surrounded by hydrophobic
nanostructures, bio-molecular probes of at least one type for
detecting molecules of a target biomarker to functionalize the
sensing surfaces of the microarray of hydrophilic islands. The
method includes receiving, over the functionalized sensing surfaces
of the microarray of hydrophilic islands, droplets of a biomarker
solution that includes the biomarker molecules to form an array of
droplets of the biomarker solutions on the functionalized sensing
surfaces. The method includes receiving over the array of droplets
of the biomarker solutions a layer of water-immiscible-liquid to
encapsulate the array of droplets of the biomarker solutions to
form water-immiscible-liquid encapsulated reaction chambers (e.g.,
nano or microliter volume) for controlling a reaction between the
target biomarker molecules and the bio-molecular probes. In one
embodiment, the water-immiscible-liquid includes oil. In one
embodiment, each droplet is nanoliter or less.
[0010] The method can be implemented in various ways to include one
or more of the following features. The bio-molecular probes of at
least one type can include DNA probes, and the molecules of
biomarkers can include nucleic acids. The DNA probes can include
DNA oligonucleotides. Receiving the layer of
water-immiscible-liquid to encapsulate the array of droplets of the
biomarker solutions to form water-immiscible-liquid encapsulated
reaction chambers (e.g., nano or microliter volume) for controlling
a reaction between the target biomarker molecules and the
bio-molecular probes can include facilitating hybridization of the
biomarker molecules with the DNA probes within the reaction
chambers (e.g., nano or microliter volume). The method can include
receiving labeling materials to within the reaction chambers (e.g.,
nano or microliter volume) to label bio-molecular probe attached
biomarker molecules formed responsive to the controlled reaction.
The labeling materials can include quantum-dots. The nucleic acids
can include DNA, RNA or miRNA-based nucleic acids. The sensing
areas can include a layer of silicon oxide (SiO2). The
super-hydrophobic surface can include black silicon. The target
biomarker molecules can include fluorescently labeled biomarker
molecules to determine a concentration of the target biomarker
molecules based on a fluorescent intensity of the fluorescently
labeled biomarker molecules that react with bio-molecular probes.
In one embodiment, the water-immiscible-liquid includes oil. In one
embodiment, each droplet is nanoliter or less.
[0011] In another aspect, a technique is described for enriching
biomarker molecules within a biosensor to facilitate
ultra-sensitive detection and quantification of the biomarker
molecules without an amplification of the biomarker molecules. This
technique includes mixing a sample containing biomarker molecules
with a hybridization buffer containing DNA oligo probes to form a
biomarker solution; and dispensing droplets of the biomarker
solution onto a device containing an array of micro-islands or
microarray of islands surrounded and separated by a hydrophobic
surface. The surface of each micro-island is functionalized with a
set of DNA oligo probes, and each droplet is dispensed onto a
separate micro-island to form a droplet array of the biomarker
solution which substantially coincides with the microarray of
islands or array of micro-islands. The technique includes
evaporating the droplet array to increase a concentration of the
biomarker molecules within each of the evaporated droplets as a
result of reducing volume of the droplet. When the droplet array
has evaporated to a thin layer of the biomarker solution, the
technique includes encapsulating the droplet array in a layer of
water-immiscible-liquid to stop the evaporation and to facilitate
the biomarker molecules within the droplet array to hybridize with
the DNA oligo probes within the microarray of islands or array of
micro-islands. The technique includes attaching quantum-dots linked
reporting DNA oligos to the set of DNA oligo probes within each of
the micro-islands; and subsequently selectively removes a subset of
the hybridized reporting DNA oligos from a corresponding subset of
the DNA oligo probes which are not hybridized with the biomarker
molecules. After the removal of the subset of the reporting DNA
oligos, the number of the quantum-dots linked reporting oligos
within each of the micro-islands is substantially equal to the
number of the hybridized biomarker molecules within the same
micro-island. The technique includes detecting and quantifying
concentration of the hybridized biomarker molecules by either
measuring a fluorescent intensity of each micro-island caused by
the remaining quantum-dots or by counting the number of the
remaining quantum dots using an image processing software. In one
embodiment, the water-immiscible-liquid includes oil.
[0012] In another aspect, a technique is described for enriching
biomarker molecules within a biosensor to facilitate
ultra-sensitive detection and quantification of the biomarker
molecules without an amplification of the biomarker molecules. This
technique includes providing a device containing an array of
sensing micro-islands surrounded and separated by a hydrophobic
surface. The surface of each sensing micro-island is functionalized
with a set of DNA oligo probes. The technique includes mixing a
sample containing biomarker molecules with a hybridization buffer
containing DNA oligo probes to form a biomarker solution. The
technique includes dispensing droplets of the biomarker solution
onto the surface of the device such that each droplet is dispensed
onto a separate sensing micro-island to form a droplet array of the
biomarker solution which substantially coincides with the array of
sensing micro-islands. The technique includes evaporating the
droplet array to increase a concentration of the biomarker
molecules within each of the droplets as a result of reducing
volume of the droplet. When the droplet array has evaporated to a
thin layer of the biomarker solution, the technique encapsulates
the droplet array in a layer of oil to stop the evaporation and to
facilitate the biomarker molecules within the droplet array to
hybridize with the DNA oligo probes within the array of sensing
micro-islands. The technique next removes the layer of oil and the
hybridization buffer to expose the DNA oligo probes, a subset of
the DNA oligo probes are hybridized with the biomarker molecules.
The technique labels the subset of the DNA oligo probes with
quantum-dots linked reporting DNA oligos. After labeling the subset
of the DNA oligo probes, the number of the quantum-dots linked
reporting oligos within each of the sensing micro-islands is
substantially equal to the number of the hybridized biomarker
molecules within the same sensing micro-island.
[0013] In another aspect, a technique for enriching biomarker
molecules within a biosensor to facilitate ultra-sensitive
detection and quantification of the biomarker molecules without an
amplification of the biomarker molecules is disclosed. This
technique includes providing a device containing an array of
sensing micro-islands surrounded by a hydrophobic surface and
separated by a grid of pillars. The surface of each sensing
micro-island is functionalized with a set of DNA oligo probes. The
technique includes dispensing a biomarker molecules containing
solution over the device to cover the array of sensing
micro-islands and subsequently evaporates the solution over the
array of sensing micro-islands to concentrate the biomarker
molecules onto the sensing micro-islands. The technique includes
dipping the device containing the condensed biomarker molecules in
a hybridization buffer containing a suspension of quantum-dots
linked reporting DNA oligos for a specific time (e.g., 1 second) to
form an array of self-assembled nano-droplets (e.g., nanoliter
volume or less) to immerse the concentrated biomarker molecules on
the sensing micro-islands. The technique includes encapsulating the
nano-droplets (e.g., nanoliter volume or less) with a layer of
water-immiscible-liquid to form an array of hybridization reaction
chambers (e.g., nano or microliter volume), which facilitate
hybridization both between the biomarker molecules and the DNA
oligo probes and between the DNA oligo probes and the quantum-dots
linked reporting DNA oligos. The technique includes selectively
removing a subset of the hybridized reporting DNA oligos from a
corresponding subset of the DNA oligo probes which are not
hybridized with the biomarker molecules. After the removal of the
subset of the reporting DNA oligos, the number of the quantum-dots
linked reporting oligos within each of the hydrophilic islands is
substantially equal to the number of the hybridized biomarker
molecules within the same hydrophilic island. The technique
includes detecting and quantifying concentration of the hybridized
biomarker molecules by either measuring a fluorescent intensity of
each micro-island caused by the remaining quantum-dots or by
counting the number of the remaining quantum dots using an image
processing software. In one embodiment, the water-immiscible-liquid
includes oil.
[0014] In yet another aspect, a technique for enriching biomarker
molecules within a biosensor to facilitate ultra-sensitive
detection and quantification of the biomarker molecules without an
amplification of the biomarker molecules is disclosed. This
technique includes providing a device containing an array of
sensing micro-islands surrounded and separated by a hydrophobic
surface and separated by a grid of pillars. The surface of each
sensing micro-island is functionalized with a set of DNA oligo
probes. The technique includes dispensing a solution of the
biomarker molecules solubilized in a hybridization buffer over the
device to form droplets of the biomarker molecules that cover the
array of sensing micro-islands. The technique includes evaporating
the droplets of the biomarker molecules, and while evaporating,
each of the droplets of the biomarker molecules realigns with a
corresponding hydrophilic sensing island in the array of
hydrophilic sensing islands, reduces in size, concentrates, and
become dry. The technique includes dipping the device containing
the condensed droplets in water for a specific time to form an
array of self-assembled nano-droplets (e.g., nanoliter volume or
less) to immerse the concentrated biomarker molecules on the
sensing micro-islands. The technique includes encapsulating the
array of nano-droplets (e.g., nanoliter volume or less) with a
layer of water-immiscible-liquid to form an array of hybridization
chambers (e.g., nano or microliter volume), which facilitate
hybridization between the biomarker molecules and the DNA oligo
probes. The technique includes removing the layer of
water-immiscible-liquid to expose the DNA oligo probes, and a
subset of the DNA oligo probes are hybridized with the biomarker
molecules. The technique includes labeling the subset of the DNA
oligo probes with quantum-dots linked reporting DNA oligos. After
labeling the subset of the DNA oligo probes, the number of the
quantum-dots linked reporting oligos within each of the sensing
micro-islands is substantially equal to the number of the
hybridized biomarker molecules within the same sensing
micro-island. The technique includes detecting and quantifying
concentration of the hybridized biomarker molecules by either
measuring a fluorescent intensity of each micro-island caused by
the remaining quantum-dots or by counting the number of the
remaining quantum dots using an image processing software. In one
embodiment, the water-immiscible-liquid includes oil.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1A shows a schematic illustrating an exemplary process
of dispensing nucleic acid (e.g., miRNA)-containing droplets onto
microarray of island (or micro-islands) with specific DNA oligo
probes.
[0016] FIG. 1B show a schematic illustrating an exemplary nucleic
acid enrichment process by evaporating the sample droplets.
[0017] FIG. 1C show a schematic illustrating an exemplary process
of encapsulating the thin layer of nucleic acid enriched liquid in
oil to form highly efficient hybridization chambers.
[0018] FIG. 1D shows a schematic illustrating an exemplary process
of hybridizing nucleic acids (e.g., miRNAs) with the specific
probes on each of the microarray of island (or micro-islands).
[0019] FIG. 1E shows a schematic illustrating an exemplary process
of attaching quantum-dots linked reporter oligos to all the
probes.
[0020] FIG. 1F shows a schematic illustrating an exemplary process
of using the stacking effect to selectively remove those reporter
oligos from a subset of probes which did not hybridize with the
target nucleic acids.
[0021] FIG. 2A shows a photograph of an exemplary droplet array
formed on the exemplary black Si template.
[0022] FIG. 2B shows a scanning electron microscopy (SEM)
micrograph of an exemplary black Si region on the exemplary black
Si template.
[0023] FIG. 2C shows a schematic illustration of a micro droplet
positioned on the exemplary black Si template.
[0024] FIG. 2D shows a photograph of the droplet on the exemplary
black Si template with a contact angle of about 155 degrees.
[0025] FIG. 2E shows an image demonstrating sample enrichment by
evaporating the droplets.
[0026] FIG. 3A shows a photographic image of a droplet array
dispensed on black Si template without oil encapsulation.
[0027] FIG. 3B shows a photographic image of the droplet array in
an oil encapsulation after the droplet volume is reduced to about
50% of the original volume.
[0028] FIG. 3C shows a photographic image of the droplet array in
an oil encapsulation when the droplets are nearly completely
evaporated.
[0029] FIG. 4A shows an exemplary design of a DNA probe (SEQ ID NO:
1) and Q-dot linked reporter oligo (SEQ ID NO: 2).
[0030] FIG. 4B shows an exemplary device schematic for detection
and quantification of different miRNAs and the process of using the
stacking effect to selectively remove the Q-dot linked reporter
oligos from DNA probes without hybridized nucleic acid.
[0031] FIG. 4C shows an exemplary plot of anticipated
time-dependent fluorescent signals from each specific miRNA marker
shown in FIG. 4B and the signal from negative control.
[0032] FIG. 5A presents a flowchart illustrating an exemplary
process for enriching biomarker molecules, such as DNAs and RNAs
within a biosensor to facilitate ultra-sensitive detection and
quantification of the biomarker molecules without an amplification
of the biomarker molecules.
[0033] FIG. 5B presents a flowchart illustrating an exemplary
process performed by a biosensor for enriching biomarker molecules,
such as DNAs and RNAs to facilitate ultra-sensitive detection and
quantification of the biomarker molecules without an amplification
of the biomarker molecules.
[0034] FIG. 6 shows an exemplary device for forming a droplet
array.
[0035] FIG. 7 shows schematics illustrating an exemplary workflow
of a biosensor based on the device in FIG. 6 for bio-molecules,
such as nucleic acid detection.
[0036] FIG. 8 illustrates an exemplary fabrication process of a
device for bio-molecules detection including a microarray of
islands or micro-island array for evaporating droplets.
[0037] FIG. 9 shows schematics of an exemplary nucleic acids
detection procedure on the hydrophilic surface of the bio-molecules
detection device from FIG. 8.
[0038] FIG. 10 shows SEM images of nanopillars fabricated using the
DRIE process with 10(a) showing a 45.degree. degree view; 10(b)
showing a top view; and 10(c) showing an interface between an
hydrophilic island and the nanopillars.
[0039] FIG. 11 shows the evolution of an evaporating water droplet
on a SiO.sub.2 patterned black silicon template (11(a)-11(c)
photographic images of the droplet at evaporation time of 1 min, 20
mins, and 40 mins; 11(d) micrograph of the droplet at evaporation
time of 50 mins; 11(e) a schematic of the evolving shape of the
droplet at 1 min, 20 mins, 40 mins, 50 mins, and 53 mins; and 11(f)
contact angle and contact diameter dependence on the evaporation
time).
[0040] FIG. 12 shows photographic images illustrating a process
when a sample droplet self-aligns with the SiO.sub.2 island during
evaporation: (a) 1 minute; (b) 15 minutes; (c) 35 minutes; and (d)
45 minutes.
[0041] FIG. 13 shows: (a) a bright view image of a clean SiO.sub.2
island surrounded by black silicon after the microfabrication
process; (b) a fluorescent image of the FITC labeled DNA dried on
the SiO.sub.2 island; and (c) detected fluorescence intensity of
FITC labeled DNA dried on the SiO.sub.2 island.
[0042] FIG. 14 shows an obtained relationship between the detected
number of streptavidin-biotin binding and the concentration of
streptavidin in the sample solution.
[0043] FIG. 15 shows: (a) a dependence plot of the number of
hybridized targets and the concentration of target molecules in the
sample; (b-d) the processed images of visualized quantum dots with
a target concentration of 100 fM, 1 pM, and 10 pM,
respectively.
[0044] FIG. 16A shows putting miRNA containing RNAse-free deionized
water over microarray of islands or micro-islands functionalized
with specific DNA oligo probes.
[0045] FIG. 16B shows water is evaporated and miRNAs are condensed
to microarray of islands or micro-islands.
[0046] FIG. 16C shows dipping the device into hybridization buffer
to form self-assembled nano-droplets (e.g., nanoliter volume or
less). The hybridization buffer also contains quantum dots linked
with DNA oligos.
[0047] FIG. 16D shows oil encapsulation of the nano-droplets (e.g.,
nanoliter volume or less) to form hybridization chambers.
[0048] FIG. 16E shows miRNAs and quantum-dots linked reporter
oligos are hybridized with DNA probes on each island.
[0049] FIG. 16F shows using the stacking effect to selectively
remove reporter oligos from probes without miRNA hybridization.
[0050] FIG. 17A shows photographic image of a droplet array
self-assembled on SiO.sub.2 islands surrounded by black
silicon.
[0051] FIG. 17B shows an SEM micrograph of the black Si area.
[0052] FIG. 17C shows sample enrichment from the evaporating
droplets, wherein the fluorescently labeled DNA oligos are
condensed onto a 125 .mu.m diameter hydrophilic surface area.
[0053] FIG. 18A shows an example of self-assembled nano-droplets
(e.g., nanoliter volume or less) after dipping the miRNA condensed
sample into hybridization buffer.
[0054] FIG. 18B shows an example of oil encapsulated nano-droplet
(e.g., nanoliter volume or less) hybridization cambers.
[0055] FIG. 18C shows fluorescence from Q-dots to show the
formation of miRNA/DNA duplex within the reaction chambers.
[0056] FIG. 19 shows (a) greatly reduced hybridization time with
the oil-encapsulated nano-droplet (e.g., nanoliter volume or less)
hybridization chamber; and (b) simulated time dependent DNA
hybridization efficiency with different liquid volume.
[0057] FIG. 20 shows preliminary sensitivity measurement of the
disclosed device containing synthesized miR205 and obtained based
on the process of FIGS. 16A-16F.
[0058] FIG. 21A shows an exemplary design of a DNA probe (SEQ ID
NO: 1) and Q-dot linked reporter oligo (SEQ ID NO: 2). The stacking
effect produces a melting temperature difference for the reporter
oligo between miRNA hybridized and unhybridized probes.
[0059] FIG. 21B shows an exemplary device schematic for detection
and quantification of different miR cancer markers and the on-chip
negative control, and the process of using the stacking effect to
selectively remove the Q-dot linked reporter oligos from probes
without hybridized target miRNAs.
[0060] FIG. 21C shows an exemplary plot of anticipated
time-dependent fluorescent signals from each specific miR cancer
marker shown in FIG. 21B and the control signal.
[0061] FIG. 22A shows a 2.times.2 SiO.sub.2 array device with
circular SiO.sub.2 patterns surrounded by super-hydrophobic black
silicon.
[0062] FIG. 22B shows DNA probes are chemically cross-linked to
SiO2 pattern surfaces.
[0063] FIG. 22C shows DNA targets solubilized in diluted
hybridization buffer are added onto SiO2 surface and allowed to
evaporate.
[0064] FIG. 22D shows that while evaporating, the target solution
droplet realigns itself onto the SiO.sub.2 pattern, reduces in
size, concentrates, and dries completely.
[0065] FIG. 22E shows the device is dipped in water for a specific
time and removed, excess water escapes the super-hydrophobic black
silicon surface, leaving a nano-liter droplet on each SiO.sub.2
pattern.
[0066] FIG. 22F shows that oil is added onto the nano-liter
droplets, which stops evaporation, and the encapsulated nano-liter
droplets undergo hybridization between DNA probes and targets.
[0067] FIG. 22G shows the DNA probe-target duplexes are labeled
with quantum dots for observation under fluorescent microscope.
[0068] FIG. 23A shows top and side view of the 400 micron diameter
circular pattern prior to water immersion.
[0069] FIG. 23B shows top and side view of the nano-liter droplet
pattern after 1 second water immersion and removal having a
dome-shaped.
[0070] FIG. 23C shows parallel and 45.degree. views of an array of
nano-droplets (e.g., nanoliter volume or less) immersed in oil.
[0071] FIG. 24 shows DNA retained on SiO.sub.2 surface after drying
from water immersion at different immersion lengths of 0, 1, 2, 3,
and 13 seconds, respectively.
[0072] FIG. 25A shows 1 ml to 5 .mu.l enrichment achieved by
evaporating target DNA solution at 95.degree. C. while Teflon mold
restricts solution above the SiO.sub.2 patterns.
[0073] FIG. 25B shows 5 .mu.l to 5 nl enrichment achieved by
evaporating target DNA solution at 50.degree. C., followed by
nano-liter droplet self-assembly described in FIG. 22E.
[0074] FIG. 25C shows that 1 ml to 5 nl enrichment results in a
linear sensitivity curve ranging from 50 aM to 500 fM.
[0075] FIG. 25D shows that 5 .mu.l to 5 nl enrichment results in a
linear sensitivity curve ranging from 10 fM to 100 pM.
[0076] FIG. 26 shows hybridization completion rate vs.
hybridization time within the nano-droplets (e.g., nanoliter volume
or less).
DETAILED DESCRIPTION
Introduction and Overview
[0077] Identification of nucleic acid markers such as DNA, RNA,
specially miRNAs from blood, biofluids, and cerebrospinal fluid
(CSF) can be achieved by sequencing, quantitative-PCR, and liquid
chromatography-mass spectrometry analysis. However, these
techniques require sample enrichment via amplification, which can
incur significant burden in time and cost. For example,
polymerase-based amplification has to be accomplished by thermal
cycling and enzymatic reactions, which is not practical for the
purpose of time-sensitive clinical diagnosis. In addition,
enzymatic amplification introduces inevitable bias toward specific
sequences that greatly compromises the accuracy of the diagnosis.
The disclosed technology provides advantages over conventional
techniques and devices for nucleic acid detection and
enrichment.
[0078] Blood and biofluids contain many biomolecules, including
proteins, DNAs, and RNAs, that can be used as biomarkers for
disease diagnosis. However, low concentration levels of these
biomarkers often make accurate and rapid detection challenging. For
instance, one needs to detect circulating miRNAs at concentrations
as low as 10 to 100 femtomolar (fM) for cancers, traumatic brain
injuries, cardiovascular diseases, etc. Most of the surface
reaction-based biosensors and DNA microarrays have a detection
limit of picomolar (pM), even when the most advanced detection
technologies are used (e.g., with fluorescence, current, or SPR).
The detection sensitivity is largely limited by the diffusion
process when the concentration of the target biomarkers drops to fM
range because the flux of diffusion is lowered by the decreasing
concentration. The technology disclosed in this patent document
includes detection techniques that can potentially overcome the
diffusion limit to allow accurate and rapid detection of low
concentrations of biomarkers.
[0079] Evaporating droplets (e.g., droplets that are nanoliter or
less) may be used to enrich target molecules. In one
implementation, a droplet device is used to concentrate DNAs by
1.times.10.sup.4 fold. However, the detection position and the
sensing area are difficult to control for reproducible performance.
Also the dried DNAs resulted from this technique are difficult to
be identified from the background noise. In another implementing, a
microchip is used where the evaporation of DNA droplets takes place
simultaneously with hybridization. In this approach, the salt
concentration continues to increase with the shrinking volume of
the droplet during the hybridization process, making the control of
hybridization conditions, especially the salt concentration,
temperature, and reaction time, rather difficult. Moreover, the
sample may be dried up before the reaction is complete. These
factors have limited the detection sensitivity of such device to
around 100 pM. In yet another implementation, a two-stage
enrichment device is developed where the target nucleic acids are
first captured by microbeads and then dried for fluorescent
detection. By separating the molecular enrichment step from the
detection step, this approach has improved the overall device
performance to pM sensitivity. However, this approach still does
not enable precise control of the hybridization conditions to reach
the sensitivity required for certain point-of-care in-vitro
diagnostic applications.
[0080] Techniques, devices, and systems are disclosed for enriching
and detecting a sample target molecule, that includes nucleic acids
(also referred to as "the disclosed technology"). In some
implementations, the disclosed technology provides a chip-based
device that can concentrate nucleic acids in a fluidic sample
on-chip. Such a chip-based device can be used to dramatically lower
the miRNA detection limit below existing technologies to reach 1
femtomolar level. The disclosed techniques can be implemented
without using any enzymatic reagents, and thus presents itself as a
viable technology for miRNA-based point-of-care and clinical
diagnosis solution to date.
[0081] The disclosed technology includes an water-immiscible-liquid
(e.g., oil)-encapsulated evaporating droplet (e.g., droplet that is
nanoliter or less) array on a microchip that can detect molecules
at a concentration of fM range. Templates having surface properties
that support the droplets have been engineered to allow the
droplets to be self-aligned with the sensing areas to facilitate
the binding or hybridization process. The disclosed technology
further utilizes evaporation for pre-concentration of the detection
targets to greatly shorten the reaction time and enhance the
detection sensitivity. In the disclosed technology, the evaporation
process of the droplets may be facilitated by the hydrophobic or
superhydrophobic surface and resulting nano-droplets (e.g.,
nanoliter volume or less) can be encapsulated by
water-immiscible-liquid (e.g., oil) drops to form stable reaction
chamber (e.g., nano or microliter volume). To further enhance the
sensitivity and specificity, a technique has been developed to have
the target enrichment and molecular detection in the same areas of
the device without intrachip or interchip sample transfer. Using
the disclosed technology, desirable droplet volumes, concentrations
of target molecules, and reaction conditions (salt concentrations,
reaction temperature, etc.) in favor of fast and sensitive
detection can be obtained. In some implementations, a linear
response over 2 orders of magnitude in target concentration can be
achieved at 10 fM for protein targets and 100 fM for miRNA mimic
oligonucleotides.
[0082] Techniques, devices, and systems of the disclosed technology
include: (1) lab-on-a-chip device architecture to perform
enrichment and detection of a sample containing nucleic acids,
without miRNA-specific label; (2) an evaporating droplet technique
to enrich the nucleic acid biomarkers such as miRNAs; (3)
water-immiscible-liquid (e.g., oil)-encapsulated reaction chambers
(e.g. nano or micro-liter volume) for efficient and reproducible
nucleic acid hybridization; and (4) detection and quantification
techniques of DNAs or RNAs using a "stacking effect".
Implementations of the disclosed technology can facilitate
achieving: (1) parallel operation with multiple samples; (2)
simultaneous detection of multiple RNA/DNA disease markers; (3)
large enrichment without amplification; and (4) femtomolar
sensitivity with high accuracy and specificity.
[0083] The disclosed technology can be used for enriching and
detection of biomarkers molecules, such as DNAs and RNAs, without
requiring any amplification steps to increase the copy number of
those molecular markers. Such devices and systems are especially
attractive to a variety of applications including, but not limited
to: gene expression analysis by estimating the copy numbers of cDNA
or mRNA transcripts present in the sample; applications in
molecular detection, nucleic acid biomarker quantification for
clinical, laboratory and point-of-care diagnostic purposes;
development to detect pathogen and infectious disease with direct
identification of endogenous sequences, including fragmental
microbial genome (DNA) and retroviral RNA sequences; capability to
quantify miRNA and other forms of circulating epigenetic marker for
early diagnosis of disease and injury; and mutational analysis of
cancer and hereditary diseases using tiling probes layout.
[0084] The disclosed lab-on-a-chip detection technique and device
can be used for early stage cancer/precancerous detection from
circulating extra-cellular vesicle (EV) encapsulated miRNAs in
blood, biofluids, or cerebrospinal fluid (CSF), allowing rapid and
accurate diagnosis at a fraction of current cost. The disclosed
detection technique is highly sensitive, reliable and requires no
amplification, thus removing any bias associated with the
amplification process. The disclosed device is compact, easy to
use, low cost, and quick in producing test results from a small
sample amount. Hence, the disclosed detection technique and device
provide a platform technology applicable to miRNA and DNA
biomarkers for various cancer types/subtypes.
[0085] In one aspect, capabilities for providing enhanced
sensitivity relative to sequencing and PCR assays are added to the
hybridization-based assays that provide advantages in amplification
free, low infrastructural cost and high-throughput capabilities.
For example, the disclosed technology can provide for a massively
parallel nano-droplet (e.g., nanoliter volume or less) microarray
system and device with detection limit as low as 50 aM. Some
embodiments of the disclosed system and device incorporate a
micro-patterned super-hydrophobic black silicon surface that
enriches nucleic acid samples by rapid evaporation and
self-assembled nano-droplet (e.g., nanoliter volume or less) array
formation. In addition of achieving an unprecedented attomolar (aM)
level sensitivity performance, some embodiments of the disclosed
system and device can also exhibit a 6 orders of linear dynamic
range and rapid hybridization time of 5 min. As a platform
technology, it can be applied to a large number of research and
clinical applications, including point-of-care disease diagnosis,
real-time pathogen detection, and microRNA based early-stage cancer
diagnosis and prognosis.
[0086] For nucleic acid detection and quantification, PCR-based
amplification assay and next generation sequencing can provide
enhanced sensitivity over other techniques such as
hybridization-based microarray. However, PCR-based assay such as
quantitative real-time PCR can only provide semi-quantitative
result. While digital PCR can potentially provide absolute
quantification, the throughput remains low. Next generation
sequencing has high infrastructural cost and is non-quantitative,
and remains a tool for discovering new DNA fragments and microRNAs.
In comparison, microarray can provide the point-of-care capability
with its amplification-free process, high throughput lacked by
PCR-based assays, and significantly lower infrastructural cost that
undermines next generation sequencing. The disclosed technology can
provide microarrays with enhanced sensitivity, high dynamic range,
and short hybridization time while providing an absolute
quantification.
[0087] Nano-particles labeling, elaborate nucleic acid probe
design, surface chemistry, electrochemistry, or microfluidic design
at the labeling and detection step of the assay can be improved.
Currently, the limit of detection from these approaches stands at
1-100 fM, and a dynamic range of 3-4 orders. In comparison to
previous attempts, the disclosed technology provides innovations in
the hybridization step prior to labeling and detection. Using the
disclosed technology, a self-assembled nano-liter droplet
microarray device has been designed to demonstrate a detecting
limit of 50 aM. In some implementations, the disclosed device
includes hydrophilic (e.g., SiO.sub.2, metal, dielectrics, or other
hydrophilic materials) patterns surrounded by micro- or
nano-patterned, hydrophobic or super-hydrophobic, black silicon
surface. Nucleic acid samples can be enriched and hybridized on
such a device via rapid evaporation followed by self-assembled
nano-droplet (e.g., nanoliter volume or less) formation. Within the
nano-liter droplets, the hybridization reaction proceeds to
completion rapidly due to the reduced volume, resulting in
accelerated hybridization time. Moreover, the precise volume
control offered by self-assembled nano-droplet (e.g., nanoliter
volume or less) array allows for a wide range of control over
enrichment ratio, resulting in higher sensitivity and extended
dynamic range. The hybridized nucleic acid samples have been
biotin-labeled and visualized by binding to quantum dots for
absolute quantification. However, the proposed platform technology
can be combined with other labeling and detection technique.
[0088] In one aspect, the disclosed technology can potentially
provide for a 200,000 fold enrichment of the sample nucleic acid,
and a self-assembled nano-liter droplet platform suitable for
massively parallel processes. Such a self-assembled nano-liter
droplet microarray system can have a detecting limit at 50 aM (50
zmol), 6 orders of linear dynamic range, and a hybridization time
of approximately 5 minutes. As a platform technology, the
nano-liter droplet microarray is suitable for point-of-care
application as it does not require sample amplification, and can be
applied to many research and clinical assays that detect and
quantify short nucleic acid.
[0089] Clinically relevant short nucleic acids biomarkers such as
viral and bacterial DNA fragments are routinely used to diagnose
infectious diseases such as tuberculosis (TB), human
immunodeficiency virus (HIV), methicillin-resistant staphylococcus
aureus (MRSA), and group B streptococcus (GBS). The other class of
clinically relevant short nucleic acid, microRNAs, are regulatory
RNAs .about.22 nucleotides in length, and can be extracted from
plasma, serum, tissue, and cells. MircoRNAs are exceptional
biomarkers for central nervous system injuries, cardiovascular
diseases, and cancer diagnosis and prognosis. Most microRNAs
studies on cancer and cardio-vascular diseases report microRNA
concentration ranging from 1 fM to 1 pM. Consequently, with a
detecting limit of 50 aM and 6 orders of linear dynamic range
without sample amplification, the disclosed system and device
provide a promising outlook for microRNA quantification,
point-of-care infectious disease diagnosis, and real-time pathogen
detection.
Exemplary Device Architecture and Process Flow
[0090] In one example, a device can handles nucleic acids (e.g.
miRNAs) in biofluids. For miRNAs that are contained within
extra-cellular vesicles or attached to Aga-protein in blood,
biofluids, or CSF, they can be extracted using standard DNA/RNA
extraction kits. The disclosed molecular enrichment and detection
devices can solve the technology bottleneck, e.g., such as for
nucleic acid biomarker enrichment and detection.
[0091] FIGS. 1A-1F show schematics illustrating an exemplary
process for enriching and detection of biomarker molecules, such as
DNAs and RNAs. FIG. 1A shows a schematic illustrating an exemplary
process of dispensing nucleic acid (e.g., miRNA)-containing
droplets onto microarray of islands or micro-islands with specific
DNA oligo probes formed on a lab-on-a-chip device 100. The
microarray of islands or micro-islands 102, each containing oligo
probes 104, are surrounded by super-hydrophobic surface. Microarray
of islands or micro-islands 102 can be hydrophilic islands. As can
be seen in FIG. 1A, the nucleic acid 106 containing sample is mixed
in diluted hybridization buffer and dispensed onto lab-on-a-chip
device 100 in forms of droplets 108.
[0092] FIG. 1B shows a schematic illustrating an exemplary nucleic
acid enrichment process by evaporating the sample droplets. As can
be seen in FIG. 1B, as sample droplets 108 evaporate on the
super-hydrophobic surface of lab-on-a-chip device 100 with
hydrophilic islands, the nucleic acid concentration increases.
[0093] FIG. 1C shows a schematic illustrating an exemplary process
of encapsulating the thin layer of nucleic acid enriched liquid in
oil to form highly efficient hybridization chambers (e.g., nano or
microliter volume). As can be seen in FIG. 1C, when sample droplets
108 are almost completely evaporated, but with a thin layer of
sample liquid remaining to covering the hydrophilic islands, oil
110 is added to cover the remaining sample droplets 108, forming a
stable microenvironment to facilitate nucleic acid hybridization
with the DNA oligo probes. In some embodiments, after the
completion of the hybridization, the process also includes removing
the layer of oil and the hybridization buffer to expose the DNA
oligo probes, as shown in FIG. 1D.
[0094] FIG. 1D shows a schematic illustrating an exemplary results
of hybridization between nucleic acids (e.g., miRNAs) 106 and DNA
probes 104 on each of the microarray of islands or micro-islands
102. In the embodiment shown, the numbers of hybridization between
nucleic acids 106 and probes 104 are, 2, 3, and 1 for the three
islands from left to right, respectively. The highly enriched
nucleic acid within the thin (e.g., 20 .mu.m) layer of the sample
liquid renders high and repeatable hybridization efficiency within
a short time (e.g., <30 minutes instead of 100 hours), as is
discussed in more details below.
[0095] FIG. 1E shows a schematic illustrating an exemplary process
of attaching quantum-dots (or "Q-dots") linked reporter or
"reporting" oligos 112 to some or substantially all the DNA probes
104. As can be seen in FIG. 1E, after washing, reporting DNA oligos
112 linked with quantum dots are introduced. In some embodiments,
the introduced reporting DNA oligos 112 have their nucleotide
sequence complementary to the first 8 nucleotides of the DNA probes
104 and hybridize with substantially all the DNA probes.
[0096] FIG. 1F shows a schematic illustrating an exemplary process
of using the stacking effect to selectively remove those reporter
oligos 112 from a subset of probes 104 which did not hybridize with
the target nucleic acids 106. In one embodiment, after washing away
extra reporting DNA oligos linked with Q-dots, the device is heated
or experiences shear stress from a relatively strong flow. Because
of the "stacking effect", the reporting oligos attached to DNA
probes hybridized with a miRNA molecule have a higher melting
temperature (T.sub.m) than the reporting oligos attached to those
DNA probes without miRNA hybridization. As can be seen in FIG. 1F,
by utilizing this stacking effect, reporting oligos linked with
Q-dots 112 stay only on those probes hybridized with the target
nucleic acids. The concentration of the specific target nucleic
acids can then be detected and quantified by, for example,
measuring the fluorescent intensity of each micro-island (due to
the remaining Q-dots) or by counting the number of quantum dots
using an image processing software.
Exemplary Techniques for Nucleic Acid Enrichment Using Evaporating
Droplets
[0097] Some disclosed techniques can be performed to enrich nucleic
acid concentrations substantially to detect and quantify low
concentrations of nucleic acids without amplification, such as the
technique based on the droplet array evaporation described above in
conjunction with FIGS. 1A-1F. Prior to performing the technique, a
template is fabricated to contain a 2-dimensional (2D) array of
hydrophilic islands surrounded by super-hydrophobic surface. FIGS.
2A-2E show images and schematics depicting an exemplary device
including a droplet array formed on hydrophilic islands surrounded
by super-hydrophobic black Si. FIG. 2 shows the effect of
enrichment using evaporating droplets.
[0098] FIG. 2A shows a photograph of an exemplary droplet array
formed on the exemplary black Si template. FIG. 2B shows a scanning
electron microscopy (SEM) micrograph of an exemplary black Si
region on the exemplary black Si template. The black Si properties
are caused by the nanostructures and the material properties of
etched Si. FIG. 2C shows a schematic illustration of a micro
droplet positioned on the exemplary black Si template. The yellow
area represents the hydrophilic island, which is surrounded by
black Si. FIG. 2D shows a photograph of the droplet on the
exemplary black Si template with a contact angle of about 155
degrees.
[0099] In one embodiment, to form the droplet array shown in FIG.
2A, nucleic acids extracted from the biofluids or blood from
patients can be mixed in highly diluted hybridization buffer and
dispensed onto the template at an initial droplet volume of 4 .mu.L
each and covering a 125 .mu.m diameter hydrophilic island on which
specific DNA probes are immobilized. In some embodiments, as the
droplets evaporate on the device surface, each droplet volume
shrinks and centers at the corresponding hydrophilic island,
eventually forming a thin layer of liquid layer (e.g., about 20
.mu.m thick) covering the hydrophilic area for a liquid volume of
around 0.4 nL, i.e., achieving 10,000.times. enrichment. The liquid
thickness can be monitored from the top of the device by monitoring
the focal length with a low magnification (10.times.) objective
lens or observed and measured from side in a setup similar to the
one for the contact angle measurement of liquid droplets. As the
nucleic acid concentration reaches the desired level determined by
the specific applications and the range of copy number of nucleic
acid intended to measure, the evaporation process may be stopped by
either oil encapsulation or by controlling the humidity of the
microenvironment. For example, if one plans to enrich the nucleic
acid from 10 fM to 100 pM (i.e., 10,000.times. sample enrichment),
one may use 10,000.times. diluted hybridization buffer to form the
initial droplet so that the optimal hybridization condition is
achieved and maintained when the volume of the droplet is reduced
to one ten thousands of the original volume. Next, fluorescently
labeled DNA oligos are added to the liquid.
[0100] FIG. 2E shows an image demonstrating sample enrichment by
evaporating the droplets. In the example shown, the fluorescently
labeled DNA oligos in a 4 .mu.L (e.g., .about.2 mm diameter)
droplet are condensed into a .about.125 .mu.m diameter hydrophilic
surface area. At the end of the enriching process, DNAs are
concentrated to the .about.125 .mu.m diameter hydrophilic surface,
and the surrounding super-hydrophobic black silicon can eliminate
the undesirable "coffee ring effect" that traps nucleic acids
during the droplet drying process.
Exemplary Oil-Encapsulated Reaction Chambers (e.g., Nano or
Microliter Volume) for Efficient miRNA Hybridization
[0101] The hybridization efficiency between the target nucleic acid
and the DNA probe is a significant factor of variation for
quantitative detection of nucleic acid (e.g., miRNA) markers. To
obtain accurate and repeatable results, the equilibrium state of
nucleic acid/DNA binding needs to be obtained. Silicone oil or
hexane can be used to encapsulate the thin layer of liquid produced
by the evaporated droplet to form a "hybridization chamber" (e.g.,
nano or microliter volume) to assure high and repeatable
hybridization efficiency.
[0102] FIGS. 3A-3C show photographic images of exemplary oil
encapsulated hybridization chambers (e.g., nano or microliter
volume). FIG. 3A shows a photographic image of a droplet array
dispensed on black Si template without oil encapsulation. FIG. 3B
shows a photographic image of the droplet array in an oil
encapsulation when the droplet volume is reduced to about 50% of
the original volume. FIG. 3C shows a photographic image of the
droplet array in an oil encapsulation when the droplets are nearly
completely evaporated, leaving a thin layer of liquid over the
hydrophilic areas. The enhanced green fluorescent intensity shows
the effects of sample concentration and accelerated hybridization.
FIGS. 3A and 3B are photographs under unfiltered white light, while
FIG. 3C is a photograph under filtered fluorescence.
[0103] The kinetics of the hybridization process can be described
by the following equations:
.differential. C .differential. t = D .differential. 2 C
.differential. x 2 x > 0 ( 1 - a ) .differential. C
.differential. t = D .differential. 2 C .differential. x 2 - C
.tau. c + KQ T 1 + KL .tau. c , x = 0 ( position of the DNA probe )
( 1 - b ) ##EQU00001##
C: target nucleic acid concentration; D: nucleic acid diffusivity;
Q.sub.T: total amount of target nucleic acid in the droplet;
.tau..sub.c: time for hybridization with the DNA probe (unit:
seconds); and K: dissociation constant of nucleic acid/DNA oligo
complex (unit: 1/sec-cm); and L: liquid thickness.
[0104] Equation (1) can be solved analytically to yield the time
dependence of hybridization efficiency, defined as the fraction of
target nucleic acids that are hybridized with the DNA probes:
.eta. ( t ) .ident. C S ( t ) Q T = [ 1 1 + K .tau. c L ] { 1 - exp
[ - Dt ( 2 L .pi. ) 2 ] } ( 2 ) ##EQU00002##
[0105] Equation (2) suggests that the time to reach equilibrium is
proportional to L.sup.2/D, provided that there exist enough number
of probes for the target nucleic acids to hybridize with, which can
be obtained in the exemplary designs. For relatively short (e.g.,
20 to 2K nt) DNAs or RNAs with D being in the order of 10.sup.-7
cm.sup.2/s, it takes longer than 100 hours for the reaction to
reach equilibrium in a typical reactor (e.g., 96-well plate) with a
liquid height of a few millimeters. By reducing the liquid
thickness to .about.20 .mu.m, the time to reach equilibrium state
can be reduced by more than 3,000 times to be as short as 5
minutes.
[0106] Secondly, the prefactor
1 1 + K .tau. c L ##EQU00003##
in Equation (2) suggests that high hybridization efficiency can be
achieved at equilibrium only if L<1/(K.tau..sub.c) (i.e., the
liquid thickness is thinner than the characteristic length
determined by the countering processes of hybridization and
dissociation). The above condition cannot be met with a large
(e.g., L=5 mm) liquid thickness in conventional well-plates designs
unless the temperature is substantially below the melting
(denaturing) temperature, thus slowing the hybridization process
further because the diffusivity in Equation (2) is also reduced
with the decreasing temperature. By reducing the liquid thickness,
the necessary condition for high miRNA hybridization efficiency:
L<1/(K.tau..sub.c) can be met, thus producing high and
repeatable nucleic acid hybridization efficiency within a short
time (e.g., <10 minutes). The allowance of using higher
hybridization temperature also improves the stringency of
hybridization, which can be especially important for miRNA
detection because many miRNAs may be different by 1 or 2
nucleotides for a typical length of around 20 nucleotides.
Exemplary on-Chip miRNA Detection Using the Stacking Effect
[0107] As mentioned previously, one way to detect and quantify the
target nucleic acids that are hybridized with the DNA oligo probes
is to use the stacking effect. For example, after nucleic acid
hybridization in the oil-encapsulated reaction chambers (e.g., nano
or microliter volume), a relatively high concentration (e.g., 10
nM) of quantum-dot linked reporter DNA oligos is introduced to
allow these reporter DNA oligos to hybridize with the DNA probes.
In some embodiments, different DNA probes that are designed to
match different target miRNAs have the same sequence for the first
8 nucleotides so that substantially all the probes can hybridize
with the Q-dot linked reporting DNA oligos, as was shown in FIG.
1E. After washing off extra Q-dot linked reporter DNA oligos, there
exist DNA probes in two possible states: probes hybridized with
reporter DNA oligos but without the target nucleic acid and probes
hybridized with both the target nucleic acid and the reporter DNA
oligos. Because of the stacking effect, the binding strength of the
DNA oligos is enhanced by the hybridized nucleic acids that are
immediately next to the DNA oligos with a nick. By introducing a
relatively strong flow that exerts shear stress to the Q-dot linked
reporter DNA oligos, one can selectively remove the Q-dot linked
reporter oligos from the unhybridized DNA probes (in terms of the
target nucleic acid) and subsequently measure the number of target
nucleic acids within each island based on the fluorescence
intensity of the remaining Q-dots.
[0108] FIGS. 4A-4C show schematics of exemplary nucleic acid
detection and quantification techniques using Q-dot linked
reporting oligo and the stacking effect. FIG. 4A shows an exemplary
design of a DNA probe and Q-dot linked reporter oligo. As mentioned
above, the stacking effect can produce a melting temperature
difference for the reporting oligo between the nucleic acid (e.g.,
miRNA) hybridized and unhybridized probes. FIG. 4B shows an
exemplary device schematic for detection and quantification of
different miRNAs and the process of using the stacking effect to
selectively remove the Q-dot linked reporter oligos from DNA probes
without hybridized nucleic acid. FIG. 4C shows an exemplary plot of
anticipated time-dependent fluorescent signals from each specific
miRNA marker shown in FIG. 4B and the signal from negative control.
As shown by the plot, a high number of hybridized target nucleic
acids in a given sample island results in a higher detected
fluorescent intensity from that sample island.
[0109] FIG. 5A presents a flowchart illustrating an exemplary
process 500 for enriching biomarker molecules, such as DNAs and
RNAs within a biosensor to facilitate ultra-sensitive detection and
quantification of the biomarker molecules without an amplification
of the biomarker molecules. The process may include mixing a sample
containing biomarker molecules, including DNAs, RNAs, miRNAs, and
other nucleic acids with a hybridization buffer containing DNA
oligo probes, thereby forming a biomarker solution (502). The
process also includes dispensing droplets of the biomarker solution
onto a device containing an array of micro-islands or microarray of
islands surrounded and separated by a hydrophobic surface (504).
The surface of each micro-island is functionalized with a set of
DNA oligo probes, and each droplet is dispensed onto a separate
micro-island to form a droplet array of the biomarker solution
which substantially coincides with the array of micro-islands or
microarray of islands The process then evaporates the droplet array
to increase a concentration of the biomarker molecules within each
of the evaporated droplets as a result of reducing volume of the
droplet (506).
[0110] After the droplet array has evaporated to a thin layer of
the biomarker solution, the process then encapsulates the droplet
array in a layer of oil to stop the evaporation and to facilitate
the biomarker molecules within the droplet array to hybridize with
the DNA oligo probes within the microarray of islands or array of
micro-islands (508). After the completion of the hybridization, the
process then removes the layer of oil and the hybridization buffer
to expose the DNA oligo probes (510). Next, the process attaches
quantum-dots linked reporting DNA oligos to the set of DNA oligo
probes within each of the microarray of islands or array of
micro-islands (512). The process then selectively removes a subset
of the hybridized reporting DNA oligos from a corresponding subset
of the DNA oligo probes which are not hybridized with the biomarker
molecules (514). Hence, after the removal of the subset of the
reporting DNA oligos, the number of the quantum-dots linked
reporting oligos within each of the microarray of islands or array
of micro-islands is substantially equal to the number of the
hybridized biomarker molecules within the same micro-island. The
process can also include detecting and quantifying concentration of
the hybridized biomarker molecules by either measuring a
fluorescent intensity of each micro-island caused by the remaining
quantum-dots or by counting the number of the remaining quantum
dots using an image processing software (516).
[0111] FIG. 5B presents a flowchart illustrating an exemplary
process 520 performed by a biosensor device for enriching biomarker
molecules, such as DNAs and RNAs to facilitate ultra-sensitive
detection and quantification of the biomarker molecules without an
amplification of the biomarker molecules. A biosensor device as
described in this patent document can perform the process 520 to
enrich and detect biomarker molecules includes receiving, by a
biosensor device including a microarray of hydrophilic islands
having sensing surfaces and surrounded by hydrophobic
nanostructures, bio-molecular probes of at least one type for
detecting molecules of a target biomarker to functionalize the
sensing surfaces of the microarray of hydrophilic islands (522).
The method includes receiving, over the functionalized sensing
surfaces of the microarray of hydrophilic islands, droplets of a
biomarker solution that includes the biomarker molecules to form an
array of droplets of the biomarker solutions on the functionalized
sensing surfaces (524). The method includes receiving over the
array of droplets of the biomarker solutions a layer of
water-immiscible-liquid to encapsulate the array of droplets of the
biomarker solutions to form water-immiscible-liquid encapsulated
reaction chambers (e.g., nano or microliter volume) for controlling
a reaction between the target biomarker molecules and the
bio-molecular probes (526). In one embodiment, the
water-immiscible-liquid includes oil. In one embodiment, each
droplet is nanoliter or less.
[0112] The method can be implemented in various ways to include one
or more of the following features. The bio-molecular probes of at
least one type can include DNA probes, and the molecules of
biomarkers can include nucleic acids. The DNA probes can include
DNA oligonucleotides. Receiving the layer of
water-immiscible-liquid to encapsulate the array of droplets of the
biomarker solutions to form water-immiscible-liquid encapsulated
reaction chambers (e.g., nano or microliter volume) for controlling
a reaction between the target biomarker molecules and the
bio-molecular probes can include facilitating hybridization of the
biomarker molecules with the DNA probes within the reaction
chambers (e.g., nano or microliter volume). The method can include
receiving labeling materials to within the reaction chambers (e.g.,
nano or microliter volume) to label bio-molecular probe attached
biomarker molecules formed responsive to the controlled reaction.
The labeling materials can include quantum-dots. The nucleic acids
can include DNA, RNA or miRNA-based nucleic acids. The sensing
areas can include a layer of silicon oxide (SiO2). The
super-hydrophobic surface can include black silicon. The target
biomarker molecules can include fluorescently labeled biomarker
molecules to determine a concentration of the target biomarker
molecules based on a fluorescent intensity of the fluorescently
labeled biomarker molecules that react with bio-molecular probes.
In one embodiment, the water-immiscible-liquid includes oil. In one
embodiment, each droplet is nanoliter or less.
Exemplary Embodiments of a Sample Enrichment Template
[0113] In some embodiments, the disclosed techniques, devices, and
systems of nucleic acid sample enrichment by evaporating droplets
can overcome the so-called "coffee ring effect." On a typical
surface, the suspended particles and macro molecules may
precipitate on the surface when the liquid droplet is reduced to a
certain size, leaving marks or stains on the surface, referred to
as "coffee ring effect." Without eliminating the coffee ring
effect, the target nucleic acids may not be condensed on the
microarray of islands or array of micro-islands where the molecular
probes are immobilized. This coffee ring effect may be caused by a
weak Marangoni flow inside the droplet when the liquid/air boundary
of the droplet moves towards the substrate. To strengthen the
Marangoni flow which drives the nucleic acids toward the center of
the droplet instead of forming the "coffee ring", various
embodiments can reduce the surface tension by adding surfactant,
using AC electrowetting to unpin the contact line as the droplet
evaporates, or creating super-hydrophobic surface. In the
discussion below, exemplary embodiments of a black silicon template
and a nanostructured polymer template are described to create the
super-hydrophobic surface. A super-hydrophobic surface can produce
a large contact angle of well over 90 degrees for droplets of
aqueous solution. In the case of black silicon and nanostructured
polydimethylsiloxane (PDMS) surfaces, the contact angle of water
droplets can be greater than 150 degrees.
[0114] In one embodiment to achieve super-hydrophobic surface, the
device template can include an array of hydrophilic micro-islands
or microarray of hydrophilic islands surrounded by
super-hydrophobic black silicon fabricated on a Si wafer. To form
black silicon, an exemplary Bosch etching process is employed in a
reactive ion etcher. The etch process includes alternating cycles
of etching (SF.sub.6) and passivation (C.sub.4F.sub.8) to form
nanopillar structures. For areas that are protected by
lithographically defined SiO.sub.2 patterns, no etching or
passivation occurs so the hydrophilic properties are preserved
after the black silicon process. The 2D array of hydrophilic
micro-island is then coated with specific DNA probes matching
specific nucleic acid targets.
[0115] In another embodiment to achieve super-hydrophobic surface,
the device template can include an array of hydrophilic
micro-islands or microarray of hydrophilic islands surrounded by
super-hydrophobic PDMS pillars formed using nanoimprinting or hot
embossing process. To obtain such a device template, an electron
beam lithographically patterned nanopillars are first formed on a
silicon wafer as the mold. For example, the diameter of the pillar
can be in a range of 50 nm to 250 nm and the center-to-center
distance of these pillars is about twice of the pillar diameter.
The pillars may be arranged in a square or hexagonal lattice or in
a semi-random fashion. Next, a "daughter mold" is formed from the
silicon master mold. Due to the nature of the imprinting process,
the daughter mold has an array of holes, complementary to the
pillars. The daughter mold may be made of Perfluoropolyether (PFPE)
Fluorolink MD700 (e.g., Solvay Solexis) as a stamp. The
complementary patterns on the stamp can be formed on the PDMS
material using standard nano-imprinting or hot embossing process.
PDMS surface by itself is hydrophobic. When such nanopatterns are
formed, the hydrophobicity increases to give an ultra large contact
angle comparable to the hydrophobicity of black silicon. To form
the hydrophilic microislands, a shadow mask can be used for
SiO.sub.2 deposition by sputtering or E-beam evaporation. The areas
covered by the SiO.sub.2 layer become hydrophilic.
[0116] Note that the above process to form the device template with
nanostructured PDMS surface can also be applied to other polymer
such as Cyclic Olefin Copolymer (COC) and Cyclic Olefin Polymer
(COP) that may be more suitable for volume production and may have
lower tendency of molecular adsorption than PDMS. Both COC and COP
have low autofluorescence so will generally not affect the
luminescence detection.
Example of Constructing an Oil-Encapsulated Nano-Droplet (e.g.,
Nanoliter Volume or Less) Array for Bio-Molecular Detection
Template Design
[0117] FIG. 6 illustrates an exemplary device 600 (i.e., the
template) for forming a droplet array. As can be seen in FIG. 6,
the surface of device/template 600 includes hydrophilic islands 602
surrounded by a hydrophobic or superhydrophobic surface 604 that
has a patterned nanostructure. On each hydrophilic island 602, one
type of molecular probes for a specific molecular target may be
immobilized. Each hydrophilic island 602 is covered with a thin
layer of SiO.sub.2 to attract the sample droplet and to anchor the
molecular probes because the SiO.sub.2 surface is compatible with
most of the surface modification protocols for biosensors. In some
designs, the SiO.sub.2 covered hydrophilic islands have a 400 .mu.m
diameter and are separated by 4 mm. Outside the SiO.sub.2 covered
areas, patterned nanostructures are formed to turn silicon into
black silicon having hydrophobic or superhydrophobic properties.
The black silicon fabrication process is adopted in the illustrated
design due to its high throughput and low cost. When forming a
droplet on a given hydrophilic island 602, the hydrophobic or
superhydrophobic surface 604 surrounding the hydrophilic island 602
allows the droplet to shrink with a minimal solid/liquid contact
area and sample loss. In some designs, an array of hydrophilic 20
islands is formed on a substrate and, if needed, the design can be
easily scaled according to the applications.
Device Architecture and Process Flow
[0118] FIG. 7 shows schematics illustrating an exemplary workflow
700 of a biosensor based on device 600 in FIG. 6 for bio-molecules
such as nucleic acid (e.g., DNAs and RNAs) detection. Workflow 700
includes six steps 7(a)-7(f). As can be seen in FIG. 7, in step
7(a), the probes having the complementary sequence to the target
bio-molecules, such as nucleic acids, are anchored on the SiO.sub.2
sensing area/islands. In specific implementation of step 7(a),
amine end-linked probes are immobilized to aldehyde-activated
SiO.sub.2 islands. In step 7(b), sample droplets of the target
bio-molecules mixed in diluted hybridization buffer are pipetted
onto the hydrophilic islands. In specific implementations of step
7(b), the target bio-molecules include streptavidin labeled miRNA
mimic oligonucleotides and the sample droplets of 4 .mu.L each are
dispensed on the template surface through a rough (visual)
alignment with the SiO.sub.2 islands. In step 7(c), the
concentration of the target bio-molecules, e.g., the miRNA mimic
oligonucleotides is increased and the volume of the sample droplet
is reduced by evaporation. In specific implementation of step 7(c),
through evaporation, the volume of each droplet is shrunk to 4
nL.
[0119] In step 7(d), a layer of oil is dispensed to encapsulate the
shrunk droplets to keep the droplet volume and the salt
concentration stable. As can be seen in step 7(d), an oil drop can
encapsulate a given shrunk droplet, thereby forming a microchamber
(e.g., a reaction chamber of microliter volume) or a nanochamber
(e.g., a reaction chamber of nanoliter volume). The layer of oil
stops the evaporation process and the hybridization reaction takes
place in controlled reaction conditions within the encapsulated
micro/nanochambers (which also referred to as a "micro/nano-droplet
reactor"). In step 7(e), the oil layer and hybridization buffer are
washed away, exposing the sample probes, some of which are
hybridized with the target nucleic acids. In step 7(f), the sample
probes (e.g., DNA duplex) are labeled with quantum dots (Q-dots)
for fluorescent detection. Finally, the immobilized target
bio-molecules, such as streptavidin labeled miRNA mimic
oligonucleotides are visualized and quantified after in-situ
labelling with quantum dots, such as streptavidin conjugated
quantum dots.
[0120] In some implementations, after step 7(d), the assay is
incubated at 50.degree. C. for 30 minutes or up to 6 hours before
washing in step 7(e). The length of incubation time does not show
obvious effect on detection sensitivity, indicating the diffusion
process may not be the sensitivity limiting factor within the
nano-droplet (e.g., nanoliter volume or less) reactors. In some
implementations, the workflow 700 of the biosensor is used for
protein detection. In such implementations, the nucleic acid
hybridization process is replaced with the protein-ligand binding
process.
Device Fabrication
[0121] As mentioned above, the device for bio-molecules detection
can be made of an array of hydrophilic SiO.sub.2 islands surrounded
by a hydrophobic or superhydrophobic surface. The device may start
on a Si substrate. The array of hydrophilic islands can be
fabricated on the Si surface using the conventional
photolithographic technique and nanopillars can be formed by deep
reactive ion etch (DRIE) over the rest of the Si area to create the
black silicon hydrophobic or superhydrophobic surface.
[0122] FIG. 8 illustrates an exemplary fabrication process 800 of a
device for bio-molecules detection including a micro-island array
for evaporating droplets. Fabrication process 800 includes steps
8(a)-8(f). As can be seen in FIG. 8, in step 8(a), a silicon wafer,
such as a mechanical grade silicon wafer is cleaned for
microfabrication. In step 8(b), a photoresist layer (e.g., negative
tone photoresist NR9-1500PY (Futurrex, USA)) is patterned on the
silicon wafer by lithography. The patterned photoresist layer
defines areas of hydrophilic islands as well as surrounding areas
of hydrophobic or superhydrophobic surface. After photoresist
patterning, SiO.sub.2 and chromium (Cr) layers are deposited on the
Si substrate by sputtering in step 8(c). In a specific
implementation, Cr and SiO.sub.2 films are deposited on the Si
wafer using a sputtering system (e.g., Denton Discovery 18, Denton
Vacuum, LLC) and the thickness of the Cr and SiO.sub.2 films are
100 nm and 120 nm, respectively. In step 8(d), a micro-island array
of SiO.sub.2/Cr dots is patterned on the silicon wafer. The
remaining photoresist is removed, e.g., by using acetone under
slight agitation.
[0123] After forming the micro-island array, a hydrophobic or
superhydrophobic surface of black Si is formed in step 8(e). In
some implementations of step 8(e), the hydrophobic or
superhydrophobic surface is formed by forming nanopillars using a
deep reactive ion etching (DRIE) process (e.g., using Plasmalab
System 100, Oxford Instruments). Unlike most top-down processes for
nanostructure formation that require definition of nanoscaled
patterns and pattern transfer, the nanopillars are formed naturally
during the deep reactive etching process. In an example process,
during the DRIE process, SF.sub.6 gas was flowed at 30 sccm during
the 8 seconds of reaction time, followed by a passivation cycle
when C.sub.4F.sub.8 gas was flowed at 50 sccm for 7 seconds. After
80 etching/passivation cycles, dense arrays of nanopillars were
formed with an average pillar height of 4.5 .mu.m. Also during the
DRIE process, those islands covered by the Cr layer were protected.
In the last step 8(f), the Cr layer over the microarray of islands
or array of micro-islands is removed by a Cr etchant to obtain the
SiO.sub.2 covered hydrophilic islands. The photograph in FIG. 8(g)
shows a fabricated device containing a 3.times.6 array of
hydrophilic islands. The optical reflectivity difference between
the array of SiO.sub.2 islands and the surrounding black Si can be
clearly observed.
Nucleic Acids Detection
[0124] The detection of bio-molecules, such as nucleic acids may be
performed with the above device obtained through the process
described in FIG. 8. FIG. 9 shows schematics of an exemplary
nucleic acids detection procedure 900 on the hydrophilic surface of
the bio-molecules detection device from FIG. 8. Detection procedure
900 includes steps 9(a)-9(e), which includes functionalizing the
SiO.sub.2 surface, immobilizing the DNA probe, and detecting the
target nucleic acids.
[0125] As can be seen in FIG. 9, in step 9(a), the device surface
is linked to aminopropyl-triethoxysilane (APTS or APTES) which
converts silanol group (SiOH) on the device surface to amine group
(NH.sub.2). The silicon atom in the APTES molecule also forms a
chemical bond with the oxygen of the hydroxyl group (OH). Next, in
step 9(b), APTES is bonded with glutaraldehyde (GTA), which is used
as a grafting agent for DNA immobilization. GTA binding can be
achieved through its aldehyde group (COH) forming a chemical bond
with the amino group of APTES, as shown in FIG. 9(b). Step (c)
shows DNA probe immobilization, wherein a given DNA oligonucleotide
probe with amine group at the 3' end is linked to a given aldehyde
group of the GTA. Next in step 9(d), target nucleic acids, such as
a DNA with biotin modification at the 3' end is hybridized with the
anchored DNA probe of a complementary sequence, forming DNA duplex.
In step 9(e), streptavidin conjugated quantum dots are bonded to
DNA duplex for visualization and quantification of the amounts of
hybridized DNA/RNA or DNA/DNA duplex.
Exemplary Results
Surface Roughness of Black Silicon
[0126] The evaporation process of droplets can be significantly
influenced by the surface roughness, hydrophobicity and contact
angle hysteresis. We examined the surface profile of the SiO.sub.2
patterned black silicon template using an environmental scanning
electron microscope (e.g., ESEM, FEI, XL30). FIG. 10 shows SEM
images of nanopillars fabricated using the DRIE process with 10(a)
showing an 45.degree. degree view; 10(b) showing a top view; and
10(c) showing an interface between an hydrophilic island and the
nanopillars. The nanopillars are .about.300 nm in diameter,
.about.300 nm in spacing and 4.5 .mu.m in height. All the scale
bars in 10(a)-10(c) are 2 micrometers.
[0127] In the illustrated example, the hydrophobic or
superhydrophobic property of the black Si is produced by the
fluoride coating resulted from the DRIE process and the increased
surface roughness. In FIG. 10(c), the SiO.sub.2 islands are around
1.5 .mu.m higher than the black silicon surface. Minimizing the
height difference between the SiO.sub.2 islands and the black
silicon surroundings facilitates reducing the adhesion of target
molecules to the sidewall of the islands while the sample droplet
solution shrinks by evaporation.
Contact Angle Measurement
[0128] Contact angles of a 4 .mu.L water droplet were measured at
25.degree. C. by the sessile-drop technique with a contact-angle
goniometer. The values reported herein were the averages three
measurements. The same instrument was used to observe evolution of
water droplets during evaporation. The contact angle of an
evaporating droplet was measured continuously until the droplet was
dried. Several droplets were observed during evaporation to assure
consistency of the data.
[0129] FIG. 11 shows the evolution of an evaporating water droplet
on a SiO.sub.2 patterned black silicon template. The static contact
angle is measured at .about.169.22.degree., suggesting the
hydrophobic or superhydrophobic nature of the black silicon
template. 11(a)-11(c) are photographic images of the droplet at
evaporation time of 1 min, 20 mins, and 40 mins; 11(d) is
micrograph of the droplet at evaporation time of 50 mins; 11(e) is
a schematic of the evolving shape of the droplet at 1 min (1), 20
mins (2), 40 mins (3), 50 mins (4), and 53 mins (5); and 11(f)
shows exemplary plots of the contact angle and contact diameter
dependence on the evaporation time. The scale bars in 11(a)-(c) are
1 mm, and the scale bar in 11(d) is 200 micrometers. As can be
observed from FIG. 11(f), before the droplet shrank toward the 400
.mu.m diameter hydrophilic island, the contact angle curve is
approximately a constant while the contact diameter curve decreases
approximately linearly. As soon as the boundary of the droplet
reached the SiO.sub.2 island, the contact angle curve drops
suddenly and the contact diameter curve of the droplet is pinned to
the boundary of the SiO.sub.2 island.
Self-Alignment Properties of Evaporating Droplet
[0130] An upright fluorescent microscope (e.g., Axio Imager, Zeiss)
was used to observe the droplet evaporation over time from the
top-view angle. A Xenon arc lamp was mounted on the microscope for
illumination. A 4 .mu.L droplet of water with diluted Rhodamine was
pipetted onto the patterned black silicon template. Because of the
SiO.sub.2 hydrophilic islands, the target droplet settled onto a
stable area when being dispensed. However, due to the large size
mismatch between the droplet and the SiO.sub.2 island, the droplet
was often misaligned with the SiO2 island even though the droplet
covered the SiO2 island. As the evaporation process proceeded, the
droplet shrank towards the center of the SiO.sub.2 island till the
contour of the droplet was aligned with the boundary of the
SiO.sub.2 island. For example, FIG. 12 shows photographic images
illustrating a process when a sample droplet self-aligns with the
SiO.sub.2 island during evaporation: (a) 1 minute; (b) 15 minutes;
(c) 35 minutes; and (d) 45 minutes. The scale bars in all images
(a)-(d) are 400 .mu.m. Through this self-alignment process, sample
droplets can be easily controlled on the black silicon template,
which greatly facilitates the droplet dispensing process and
molecular sensing process for point-of-care applications.
Fluorescently Labeled DNA Oligonucleotides Concentrated onto the
Sensor Area
[0131] To test the capability of the evaporating droplets for
sample enrichment, a 4 .mu.L droplet of FITC labelled DNA
oligonucleotides diluted in distilled water was pipetted on the
device. Solutions of progressively decreasing concentration were
examined. The droplets were dried at 37.degree. C. and investigated
under an inverted epifluorescence microscope (Eclipse TE2000U,
Nikon). After background subtraction, the average intensity over
the entire SiO.sub.2 island was analyzed using ImageJ and a custom
image analysis Matlab program.
[0132] FIG. 13 shows: (a) a bright view image of a clean SiO.sub.2
island surrounded by black silicon after the microfabrication
process; (b) a fluorescent image of the FITC labeled DNA dried on
the SiO.sub.2 island; and (c) detected fluorescence intensity of
FITC labeled DNA dried on the SiO.sub.2 island. The scale bars are
200 .mu.m. It can be observed from FIG. 12(b) that when the
fluorescently labelled DNA oligonucleotides solution was completely
dried on the SiO.sub.2 islands, the molecules were uniformly
distributed over the entire hydrophilic surface. The clean
background on the black silicon surrounding region suggests that
the sample loss due to the liquid/solid boundary movement during
evaporation was minimal. FIG. 12(c) shows that a concentration
lower than 50 fM was detectable above the background noise.
Protein Detection
[0133] For streptavidin detection, the hydrophilic islands were
pre-anchored with biotin-linked DNA oligonucleotides. The DNA
oligonucleotides sequence was: 5' Biotin-AAAAA AAAAA-amine 3' (SEQ
ID NO: 3). Target streptavidin was conjugated with quantum dots
(e.g., Qdot 525, Life technologies) for visualization. Sample
droplets (4 .mu.L each) with different concentrations of quantum
dots-streptavidin complex were spotted on the black silicon
template. The assay was incubated at 37.degree. C. to accelerate
the evaporation process. The contact area of the droplet is fixed
by the hydrophilic surface of the SiO2 island, and the height of
the droplet was monitored by a goniometer as the droplet volume
decreased by evaporation. When the height of the droplet approached
the target value, we optically zoomed in by 25.times. to closely
monitor the droplet height. As soon as the sample droplet shrank to
4 nL, a drop of silicone oil (e.g., S159-500, Fisher Chemical) was
employed to encapsulate the sample droplet and stop the
evaporation. The assay was further incubated at room temperature
for 1 hour before it was dipped in hexane solution to remove the
silicone oil. The assay was then cleaned by gentle shaking in TBST
buffer and Milli-Q water for 5 minutes and 3 minutes, respectively.
After blowing dry with nitrogen, the assay was ready for
observation.
[0134] The detection sensitivity of the evaporating droplet
microarray was tested by varying the target molecule (e.g., nucleic
acid or protein) concentration from 10 fM to 100 pM. The bound
Q-dots were quantified by using a custom Matlab program. As a
control sample, one device area has hydrophilic islands
pre-anchored with the scrambled probes, so that any quantum dots
left in those areas were due to incomplete wash or non-specific
binding. We obtained the real binding events by subtracting the
number of non-specifically bound Q-dots from the detected events
over the areas with DNA or ligand probes. The final results are
shown in FIG. 13, which shows an obtained relationship between the
detected number of streptavidin-biotin binding and the
concentration of streptavidin in the sample solution.
[0135] A linear relationship between the streptavidin concentration
and the number of streptavidin-Q-dots bound to the biotin probes
was obtained with the streptavidin concentration ranging from 10 fM
to 10 pM. For higher target concentration beyond 10 pM, the bonding
events were too dense to be resolved microscopically by the
specific image processing program used to obtain the relationship.
For streptavidin concentration lower than 10 fM, the results were
less reliable because the number of non-specific binding could be
comparable with the number of specific binding. The amount of
non-specific binding can be reduced by optimizing the washing
conditions and proper surface treatments of the SiO.sub.2 islands.
Furthermore, the variation of the measurements can be further
reduced by improved control of the droplet evaporation process
through automation.
The Detection of miRNA Mimic Oligonucleotides
[0136] An exemplary sequence of anchor probe oligonucleotides is:
5' TGCGA CCTCA GACTC CGGTG GAATG AAGGA AAAAA AAAAA-amine 3' (SEQ ID
NO: 1). The exemplary target is miRNA 205 mimic oligonucleotides
with a sequence of: 5' TCCTT CATTC CACCG GAGTC TGAGG TCGCA-biotin
3' (SEQ ID NO: 4). miRNA 205 mimic was used here because miR205 has
been reported as a specific biomarker for squamous cell lung
carcinoma. The hybridization buffer (2% BSA, 50 mM borate buffer,
0.05% sodium azide, pH 8.3) was diluted 1000 fold before the target
oligonucleotides were mixed in. Sample droplets (4 .mu.L each) of
different concentrations of miRNA 205 mimic oligonucleotides were
pipetted to the black silicon template to form micro-droplets.
After the evaporation and oil encapsulation process described
previously, the assay was incubated at 50.degree. C. to facilitate
hybridization. In the last step, streptavidin conjugated quantum
dots (1 nM) was introduced to label those hybridized DNA
duplex.
[0137] FIG. 15 shows: (a) a dependence plot of the number of
hybridized targets and the concentration of target molecules in the
sample; (b-d) the processed images of visualized quantum dots with
a target concentration of 100 fM, 1 pM, and 10 pM, respectively. As
was shown in FIG. 15(a), a linear relationship between the number
of detected hybridized target and the DNA target concentration was
obtained. Hence, we have achieved a sensitivity of 100 fM with a
dynamic range of 2 orders of magnitude. Yet in another embodiment
of the disclosed technology, which includes a similar process but
with a larger volume (1 mL) of initial DNA containing sample, a
sensitivity of 50 aM with a dynamic range of 4 orders of magnitude
has been achieved. This embodiment is discussed later on in this
patent disclosure, e.g., see FIG. 25C. By optimizing the
hybridization conditions such as the incubation temperature, DNA
probe density, and salt concentration by varying the buffer
dilution factor, the detention sensitivity is expected to be
further increased.
Discussion
[0138] A novel oil-encapsulated micro/nano-droplet (e.g.,
microliter volume or nanoliter volume or less) array reactor for
biosensing applications has been demonstrated. The disclosed design
has addressed the inherited slow, passive diffusion limitation
commonly observed during DNA hybridization or protein-ligand
binding by drastically decreasing the height of the reaction
aqueous layer. Furthermore, the design greatly enriches the
concentration of target molecules by several orders of magnitude in
a controllable manner. Specifically, this enrichment procedure does
not introduce amplification bias commonly found in thermal cycling
or reverse transcription process (i.e., the enrichment factors for
all the molecules are substantially the same and independent of the
GC contents of target DNAs). Hence, the disclosed system and
technique may be used as a hybridization platform for direct
detection of molecular markers of low abundance without requiring
the enzymatic amplification process such as PCR, and offers a
cost-effective, fast solution for point-of-care in-vitro
diagnosis.
[0139] Embodiments of the oil-encapsulated evaporating-droplet
molecular-detector platform are based on the fabrication of
hydrophilic islands surrounded by a hydrophobic or superhydrophobic
surface. The hydrophobic or superhydrophobic surface yields very
large contact angle (.about.160 degrees) and eliminates the coffee
ring effect caused by the receding boundary of the droplet. Black
silicon is chosen to form the hydrophobic or superhydrophobic
surface because the nanopillars that give black silicon its optical
and hydrophilic properties are formed naturally during the deep
reactive etching to form the black silicon, thereby avoiding the
slow and expensive steps of fabricating nanopatterns over a large
area.
[0140] In some implementations, protocols have been developed to
precisely control the evaporation process. Goniometer was used to
closely monitor the evolution of the droplets during evaporation.
Oil encapsulation terminated the evaporation process and formed a
stable environment for the micro/nano-droplet (e.g., microliter
volume or nanoliter volume or less) reactor without being affected
by the outside environment such as humidity. Preliminary data has
shown a detection sensitivity of 10 fM for streptavidin as a
protein target and <0.1 fM (e.g., 50 aM) for miRNA mimic
oligonucleotides. A linear response was obtained for a
concentration range spanning nearly 4 orders of magnitude. The
detection sensitivity may be further enhanced by optimizing the
hybridization conditions and reducing the diameters of hydrophilic
islands. Furthermore, the device architecture can be easily scaled
to increase the throughput and miniaturized footprint to support
various molecular detection purposes desirable for point-of-care
applications.
Exemplary Techniques and Devices for Glioblastoma Diagnosis
[0141] Glioblastoma can be used to assess the disclosed technology
because it is a dominant form of brain tumor. Detection at an early
stage provides significant benefits for intervention options to
improve the prognosis outcome. Currently, glioblastoma is primarily
diagnosed by CT, MRI and pathological examination of biopsy. Lack
of a robust molecular marker has prevented a timely and effective
diagnosis solution to this day. Recently, researchers have
identified the presence of microRNA-21 (miR-21) in the
extra-cellular vesicles (EVs) as a unique molecular signature
secreted by glioblastoma cells. Utilizing miRNAs as oncogenic
marker has sometimes raised concerns over reliability of
normalization standards. To address this issue, an EV-based
normalization technique has been developed, accompanied by a
minimum copy number cut-off protocol without the need for
controversial "reference transcripts" such as GAPDH, 18S rRNA, and
hsa-miR-103. Based on the absolute number of miR-21 per EV particle
assessment, the disclosed technique has demonstrated the capability
to quantitatively distinguish between cerebrospinal fluid (CSF)
derived from glioblastoma and from non-oncologic patients.
[0142] Based on the concept of utilizing EV-associated miR-21 in
combination with potentially additional miRNAs as a biomarker panel
(e.g. miR-92b, miR-128 and miR-192), an in-vitro platform has been
developed for reliable and accurate detection of miRNAs in CSF for
early prognostication of glioblastoma. The disclosed lab-on-a-chip
device in this patent document can be used to enrich, detect, and
quantify specific miRNA markers from CSF. As a platform technology,
it can be applied to a large number of other diseases beyond
glioblastoma diagnosis, including lung, pancreatic, kidney, breast,
liver cancer etc., as well as cardiac vascular diseases (CVDs) and
immune disorders. Moreover, the disclosed platform is expected to
be highly quantitative and more sensitive to the current
nucleic-acid based diagnosis, and allows rapid, early detection at
less than 1/10 of today's cost using a device that is 1/10 of the
cost of image-based systems. Table 1 shows comparisons between some
existing and emerging technology platforms and the disclosed
nucleic acid/miRNA technology.
TABLE-US-00001 TABLE 1 Comparisons with current and emerging
nucleic acid/miRNA technology platforms Processing Consumable time
(post Detection Sample Instrument and Reagent Technology
Manufacturer extraction) limit volume Amplification cost cost qPCR
Life Tech., 2 hr + pM 15-100 Required $20K $100-$200/96 BioRad,
Perkin uL/reaction well plate Elmer Digital PCR Formulatrix, 2 hr+
pM 10-100 Required $100K $100-$200/96 Life Tech uL/reaction well
plate Sequencing Illumina, Roche 2-7 days pM 10-50 Required
$50K-$700K $5,000 per 454, Life Tech ul/reaction flowcell This
Patent UCSD/Compliance 30 min 0.1 fM No Not required <$5K
<$20 per 8 Disclosure Decisions limitation samples
Overall Architecture Design and Concepts
[0143] The disclosed technology includes (a) lab-on-a-chip device
architecture to perform enrichment and detection without
miRNA-specific label, (b) an evaporating droplet material to enrich
miRNAs, (c) oil-encapsulated reaction chambers (e.g., nano or
microliter volume) for efficient and reproducible miRNA
hybridization, and (d) detection and quantification of miRNAs using
the "stacking effect". Implementations of the disclosed technology
can facilitate achieving: parallel operation with multiple (>8)
samples, simultaneous detection of multiple RNA/DNA cancer markers,
large enrichment without amplification, and sub-femtomolar
sensitivity with high accuracy and specificity.
[0144] In some implementations, the disclosed device handles miRNAs
(or any nucleic acid) cancer markers that are extracted from either
extra-cellular vesicles or Aga-protein in blood, biofluids, or CSF.
Specifically, the platform will be validated with short nucleic
acid oligos spiked in the nuclease-free water identical to the
miR-21 sequence extracted by the mirVANA kit (Life Technologies)
optimized for purification of RNA species <200 bps. The protocol
is proven and cost effective, and can be readily applicable to
miRNA extraction from CSF for clinical samples.
[0145] The disclosed device includes an array of hydrophilic
micro-islands or microarray of hydrophilic islands surrounded by
super hydrophobic black silicon. The hydrophilic islands includes
SiO.sub.2 coated Si with DNA probes that are complimentary to the
miR targets.
[0146] FIGS. 16A-16F summarizes an exemplary device design and
operation process flow. Note that FIGS. 16A-16F provide a different
enrichment process from the enrichment process described in
conjunction with FIGS. 1A-1F.
[0147] More specifically, FIG. 16A shows putting miRNA containing
RNAse-free deionized water over micro-islands functionalized with
specific DNA oligo probes. The microarray of islands or array of
micro-islands are surrounded by super-hydrophobic surface formed by
black silicon and separated by Teflon-coated grids. The extracted
miR sample from 0.1-1 mL CSF simulant from the mirVANA kit is
suspended in RNase free deionized water over a Teflon grid that
matches the patterns of the microislands. The device is heated to
95.degree. C. to accelerate water evaporation. FIG. 16B shows water
is evaporated and miRNAs are condensed to the microarray of islands
or array of micro-islands. As the liquid is evaporated to become
droplets (10-30 .mu.L), the temperature is reduced from 95.degree.
C. to 60.degree. C. to assure the liquid remains in the Cassie
state (i.e. slippery droplet over the black Si) instead of in the
Wenzel state (i.e. droplet pinned to the black Si). This process
leads to miRNA condensation on the microislands when the liquid is
completely dried out.
[0148] Next the entire device that contains the condensed miRNAs is
dipped in the hybridization buffer with suspension of quantum-dot
conjugated reporter DNAs for detection using the stacking effect
(described below). FIG. 16C shows dipping device into hybridization
buffer to form self-assembled nano-droplets (e.g., nanoliter volume
or less), the hybridization buffer also contains quantum dots
linked with DNA oligos. Because of the properties from surface
energy engineering, a uniform array of nano-droplets (around 0.2 nL
each) is formed as soon as the wafer is removed from the
buffer.
[0149] Next, a layer of oil is applied to encapsulate the
nano-droplets (e.g., nanoliter volume or less), forming an array of
stable, well-controlled hybridization chambers (e.g., nano or
microliter volume). FIG. 16D shows oil encapsulation of the
droplets to form hybridization chambers (e.g., nano or microliter
volume). Due to the small volume of the hybridization chamber
(e.g., nano or microliter volume) and the isolation of the reaction
chambers (e.g., nano or microliter volume) from the influences of
outside environments (humidity, edge effect, etc.), hybridization
between the target miRNAs and the complementary probes and the
Q-dot conjugated reporting DNAs occur at high efficiency, resulting
in significant improvements in sensitivity and hybridization time
(from >10 hours in a 96-well plate to <10 minutes). In some
embodiments, after the completion of the hybridization, the process
also includes removing the layer of oil and the hybridization
buffer to expose the DNA oligo probes, as shown in FIG. 16E. FIG.
16E shows miRNAs and quantum-dots linked reporter oligos are
hybridized with DNA probes on each island.
[0150] FIG. 16F shows using the stacking effect to selectively
remove reporter oligos from probes without miRNA hybridization.
After washing away extra reporting DNA oligos linked with Q-dots,
the device is heated or experiences shear stress from a relatively
strong flow. Because of the "stacking effect", the reporting oligos
attached to DNA probes hybridized with a miRNA molecule have a
higher melting temperature (Tm) than the reporting oligos attached
to those DNA probes without miRNA hybridization. Quantum dots can
be retained on those miRNA hybridized probes but not on those
probes without miRNA hybridization. The concentration of the
specific miRNA can then be obtained by measuring the fluorescent
intensity of each micro-island or by counting the number of quantum
dots using software such as ImageJ. By detecting the Q-dot
fluorescent intensity, each miRNA cancer marker can be detected and
quantified.
miRNA Enrichment Using Evaporating Droplets
[0151] To detect and quantify miRNAs without amplification, it is
necessary to enrich the miRNA concentration significantly. This
disclosed technology in conjunction with FIGS. 16A-16F provides an
effective, repeatable approach to enrich the miRNA concentration by
up to 500,000 times, which may be calculated from the initial
(spiked) miRNA concentration in CSF simulant to the final miRNA
concentration within the oil-encapsulated nano-droplet (e.g.,
nanoliter volume or less) hybridization chambers. The enrichment
factor is achieved from the ratio of initial sample volume (0.1-1
mL) and the final volume of the nano-droplet (200 pL per droplet),
giving rise to an enrichment factor of >500,000 assuming no miR
loss in the process. Because of the hydrophobicity or
superhydrophobicity of black silicon surrounding the hydrophilic
islands to suppress the coffee-ring effect, minimum sample loss is
expected.
[0152] FIGS. 17A-17C show photographic images of nucleic acid
enrichment on hydrophilic islands surrounded by super-hydrophobic
black Si. FIGS. 17A-17C may be used to prove the concept of sample
enrichment using evaporating droplets. In the examples shown,
fluorescently labeled DNA oligos were added to the liquid. FIG. 17A
shows photographic image of a droplet array self-assembled on
SiO.sub.2 islands surrounded by black silicon. FIG. 17B shows an
SEM micrograph of the black Si area. The black Si properties are
caused by the nanostructures and the material properties of etched
Si. FIG. 17B shows sample enrichment from the evaporating droplets,
wherein the fluorescently labeled DNA oligos are condensed onto a
125 .mu.m diameter hydrophilic surface area. As can be seen in FIG.
17C, at the end of the enrichment process, DNAs were concentrated
to the 125 .mu.m diameter hydrophilic surface, and the surrounding
super-hydrophobic black silicon eliminates the undesirable "coffee
ring effect" that traps nucleic acids during the droplet drying
process.
Oil-Encapsulated Reaction Chambers (e.g., Nano or Microliter
Volume) for Efficient miRNA Hybridization
[0153] The hybridization efficiency between the target nucleic acid
and the DNA probe is a significant factor of variation for
quantitative detection of nucleic acid (e.g., miRNA) markers. To
obtain accurate and repeatable results, the equilibrium state of
nucleic acid/DNA binding needs to be obtained. The following
analyses and FIGS. 18A-18C show that the proposed exemplary
oil-encapsulated nano-droplet (e.g., nanoliter volume or less)
hybridization chamber design can enable (a) drastic reduction of
reaction time from .about.10 hours to <10 minutes and (b) high
hybridization efficiency.
[0154] FIGS. 18A-18C show preliminary results following the process
flow in FIGS. 16A-16F. FIG. 18A shows an example of self-assembled
nano-droplets (e.g., nanoliter volume or less) after dipping the
miRNA condensed sample into hybridization buffer. FIG. 18B shows an
example of oil encapsulated nano-droplet (e.g., nanoliter volume or
less) hybridization cambers and FIG. 18C shows fluorescence from
Q-dots to show the formation of miRNA/DNA duplex within the
reaction chambers (e.g., nano or microliter volume).
[0155] The kinetics of the hybridization process has been described
previously by Equation (1), which can be solved analytically to
yield the time dependence of hybridization efficiency, .eta.(t) and
Equation (2). From Equation (2), the time to reach equilibrium is
proportional to L.sup.2/D provided there exist enough number of
probes for miRNA to hybridize with, which can be obtained in the
exemplary designs. For miRNAs with D being in the order of
10.sup.-7 cm.sup.2/s, it takes longer than 100 hours for the
reaction to reach equilibrium in a typical reactor (e.g. 96-well
plate) with a liquid height of millimeters. By reducing the liquid
thickness to 20 .mu.m (for 0.2 nL droplet volume), the time to
reach equilibrium state is reduced by more than 1,000 times to be
as short as 5 minutes, which was experimentally demonstrated and
results are shown in FIG. 19.
[0156] FIG. 19 shows (a) greatly reduced hybridization time with
the oil-encapsulated nano-droplet (e.g., nanoliter volume or less)
hybridization chamber; and (b) simulated time dependent DNA
hybridization efficiency with different liquid volume. With 200
.mu.L volume in most conventional devices, the reaction takes 16-24
hours to reach the equilibrium state. Using the disclosed device,
the reaction can be completed in <5 minutes.
Design and Fabrication of Black Silicon Templates for Droplet-Based
miRNA Enrichment
[0157] In some implementations, the disclosed lab-on-a-chip device
includes an array of hydrophilic micro-islands or microarray of
hydrophilic islands surrounded by super-hydrophobic black silicon
fabricated on a commercial Si wafer. To form black silicon, the
Bosch etching process can be employed in a reactive ion etcher. The
etch process includes alternating cycles of etching (SF.sub.6) and
passivation (C.sub.4F.sub.8) to form nanopillar structures. For
areas that are protected by lithographically defined SiO.sub.2
patterns, no etching or passivation occurs so the hydrophilic
properties are preserved after the black silicon forming process.
To construct a detector using the template, the 2D array of
hydrophilic micro-islands or microarray of hydrophilic islands is
coated with specific DNA probes matching specific miRNA
targets.
[0158] The miRNA extracted from CSF simulant (0.1-1 mL) is
suspended in RNAse free deionized water before being dispensed to
the lab-on-a-chip device with Teflon coated grids (see FIG. 16A).
At a concentration of 1 fM, 100 .mu.L CSF simulant contains 60,000
molecules, which gives rise to sufficient signal intensity and low
statistical noise. In some embodiments, the enrichment factor may
be characterized under different miRNA concentrations and
evaporation and wash conditions.
Development of Process for High Sensitivity (<1 fM),
Quantitative, and Specific miRNA Detection 1. miRNA Hybridization
in Oil-Encapsulated Reaction Chambers (e.g., Nano or Microliter
Volume):
[0159] As discussed previously, the hybridization efficiency and
process reproducibility can be greatly enhanced with a reduced
thickness of the liquid layer. After the miRNA condensed sample is
dipped into the hybridization buffer to form a self-assembled
droplet array, the self-assembled droplet array can be used to
characterize the following parameters: (a) the optimal composition
of the hybridization buffer (e.g., its PH value and ionic
strength); and (b) the concentration and properties (e.g., surface
charge) of Q-dot linked DNA oligos in the hybridization buffer to
minimize Q-dot residues due to incomplete wash.
[0160] FIG. 20 shows preliminary sensitivity measurement of the
disclosed device containing synthesized miR205 and obtained based
on the process of FIGS. 16A-16F. More specifically, FIG. 20 shows a
dependence plot of the number of Q-dots and the concentration of
target molecules in the sample (the upper plot); and the processed
images of visualized quantum dots with a target concentration of
100 aM, 1 fM, 10 fM, 100 fM, and 1 pM, respectively (the bottom
images). As shown by the dependence plot, a linear relationship
between the number of detected Q-dots and the target molecule
concentration is obtained. Preliminary results with miR205 have
shown that the device can obtain sensitivity of 50 aM (0.05 fM)
even though there is still room for improvement in the
reproducibility of the detection to assure repeatable (<20%
run-to-run variation) and quantitative results. In these
embodiments, quantum dots are used over fluorescent molecules
because one can count individual quantum dots that signify the
hybridization of each single miRNA molecule. This property enables
researchers to visualize and precisely measure the enrichment and
hybridization efficiency of target miRNAs, offering great
advantages during the R&D phase to optimize the device process
and protocols.
2. miRNA Detection and Quantification Using the Stacking Effect
[0161] FIGS. 21A-21C show schematics of miRNA cancer marker
detection and quantification using Q-dot linked reporting oligo and
stacking effect. FIG. 21A shows an exemplary design of a DNA probe
and Q-dot linked reporter oligo. The stacking effect produces a
melting temperature difference for the reporter oligo between miRNA
hybridized and unhybridized probes. In some implementations,
different DNA probes that are designed to match different target
miRNAs have the same sequence for the first 8 nucleotides so that
substantially all the probes can hybridize with the Q-dot linked
reporting DNA oligos, as shown in FIG. 21A.
[0162] FIG. 21B shows an exemplary device schematic for detection
and quantification of different miR cancer markers and the on-chip
negative control, and the process of using the stacking effect to
selectively remove the Q-dot linked reporter oligos from probes
without hybridized target miRNAs. After washing off extra Q-dot
linked reporter DNA oligos, the molecular probes can be in two
possible states: probes hybridized with reporter DNA but without
the target miRNAs and probes hybridized with both the target miRNAs
and the reporter DNA. Because of the stacking effect, the binding
strength of the DNA oligos is enhanced by the hybridized miRNAs
that are immediately next to the DNA oligos with a nick. Control
areas are provided where molecular probes are mismatched to any
human miRNAs. The Q-dot signals during heating or flushing can be
recorded, wherein the heating or flushing usually takes <10
minutes.
[0163] Next, it is possible to analyze how the number of quantum
dots in each area varies over time, thus can be used to produced
curves similar to those in FIG. 21C. FIG. 21C shows an exemplary
plot of anticipated time-dependent fluorescent signals from each
specific miR cancer marker shown in FIG. 21B and the control
signal. From this study, one can obtain background level due to
instrument noise, non-specific binding, and incomplete wash of
extra quantum dots. Such information will help process optimization
and signal processing to further improve the accuracy of the test.
The on-chip control signal allows for removing the effects of
non-specific binding, background noise, and residues due to
incomplete wash.
Verification of the Device Efficiency, Repeatability, Accuracy, and
Utility
[0164] In some embodiment, the disclosed device is validated by
quantitatively detecting the levels of glioblastoma miRNA marker
(miR-21) over the relevant range from 1 fM to 100 fM. The tests can
be performed using spiked synthetic miRNA oligos. The validation
process can include the following calibration steps. [0165]
Calibration of variations in miRNA enrichment factor by the droplet
process: A known amount of synthetic, fluorescently labeled miR-21
can be introduced into the sample. The emission intensity from each
run gives rise to the run-to-run variation of the miRNA enrichment
and hybridization efficiency. [0166] Calibration of the "stacking
effect": The denaturing of reporter DNA oligo from the probes is
generally a stochastic process. To produce an accurate reading of
the miRNA level, the signal will be measured with and without
synthetic miR-21. The temporal response of the emission signal with
and without target miRNAs produces data about the kinetics of the
denaturing process. These curves can be used to (a) optimize the
denaturing process control (i.e. temperature and flow rate); and
(b) find the statistical properties (mean and sigma) of the
"stacking effect". Similar curves can also be generated using qPCR
to serve as data for cross-platform comparisons. [0167] Calibration
of non-specific binding: During the process development, one can
use test probes made to have one or two nucleotides mismatch from
the target miRNA, and can test miRNAs of different GC contents. The
binding event detected from those "test probes" can help to
identify the extent of non-specific binding. In addition to
optimization of device design and experimental conditions, data
analysis and Q-dot specific image quantification techniques can be
developed to minimize uncertainties caused by non-specific
binding.
Massively Parallel Nano-Droplet (e.g., Nanoliter Volume or Less)
Microarray for Ultrasensitive Nucleic Acid Quantification
Detailed Embodiment of the Disclosed Technology
[0168] An ultra-sensitive nucleic acid quantification workflow
performed on a super-hydrophobic, nano-patterned microarray device
is disclosed. The workflow utilizes device surface
super-hydrophobicity to form self-assembled nano-droplet (e.g.,
nanoliter volume or less) array, which is massively parallel in
nature and exhibits precise droplet volume control and extreme
concentration enrichment. FIGS. 22A-22G show a process flow of
nano-liter droplet array formation, surface hybridization reaction,
and quantum dots labeling on a nano-droplet (e.g., nanoliter volume
or less) microarray device including an array of 400 um, circular
SiO.sub.2 pattern surrounded by super-hydrophobic black silicon
surfaces. FIG. 22A shows a 2.times.2 SiO.sub.2 array device with
circular SiO.sub.2 patterns surrounded by super-hydrophobic black
silicon. For nucleic acid quantification, the following steps are
implemented in the nano-droplet (e.g., nanoliter volume or less)
microarray workflow: (1) DNA probes are chemically cross-linked to
SiO2 pattern surfaces (see FIG. 22B). Next, DNA targets solubilized
in deionized water or diluted hybridization buffer are added onto
SiO2 surface to form droplets of the target solution and the target
solution is allowed to evaporate (see FIG. 22C). While evaporating,
the target solution droplet realigns itself onto the SiO.sub.2
pattern, reduces in size, concentrates, and dries completely (see
FIG. 22D).
[0169] Next, the device is dipped in hybridization buffer or water
for a specific time (e.g., 1 second) and removed, excess
hybridization buffer or water escapes the super-hydrophobic black
silicon surface, leaving a nano-liter droplet on each SiO.sub.2
pattern (see FIG. 22E). After forming the nano-droplets (e.g.,
nanoliter volume or less) on the device, oil is added onto the
nano-liter droplets, which stops evaporation, and the encapsulated
nano-liter droplets undergo hybridization between DNA probes and
targets (see FIG. 22F). In some embodiments, the device is immersed
in oil and heated to undergo target-probe hybridization within the
oil-encapsulated nano-droplets (e.g., nanoliter volume or less).
Subsequently, the device is removed from oil and the DNA
probe-target duplexes are labeled with quantum dots for observation
under fluorescent microscope (see FIG. 22G). In some embodiments,
milk protein was added to prevent non-specific binding.
[0170] To demonstrate the massively parallel nature of the
disclosed nano-droplets (e.g., nanoliter volume or less)
microarray, data are generated on droplet volume, droplet volume
variance, and nucleic acid retention resulted from the disclosed
workflow. During the proposed workflow, after drying DNA target
solution on SiO.sub.2 pattern, the device is dipped in water or
hybridization buffer to form nano-droplets (e.g., nanoliter volume
or less) on each pattern. The underlining principal of this
self-assembled process can be attributed to black silicon's
super-hydrophobicity, i.e., the black silicon-water adhesion force
is far less than water cohesion force. On the other hand, SiO.sub.2
is hydrophilic and the SiO.sub.2-water adhesion force is greater
than water cohesion force. During dipping, water adheres onto
SiO.sub.2 surfaces and escapes from the black silicon surface, thus
forming many nano-droplets (e.g., nanoliter volume or less). In
some implementations, the nano-droplet (e.g., nanoliter volume or
less) size is governed primarily by SiO.sub.2 pattern size and
contact angle of the water-SiO.sub.2 interface.
[0171] FIGS. 23A-23C show characterization of an exemplary
nano-liter droplet array obtained using the workflow shown in FIGS.
22A-22G. FIG. 23A shows top and side view of the .about.400 micron
diameter circular patterns prior to water immersion. FIG. 23B shows
top and side view of the nano-liter droplet after 1 second water or
hybridization buffer immersion and removal having a dome-shaped
pattern. The nano-droplet (e.g., nanoliter volume or less) is
formed at a height of .about.82 micron and a water contact angle on
the SiO.sub.2 surface of .about.43.5.degree.. FIG. 23C shows
parallel and 45.degree. views of an array of nano-droplets (e.g.,
nanoliter volume or less) immersed in oil.
[0172] In the embodiments shown in FIG. 23B, the geometry of water
droplet resembles a spherical cap with average height of .about.82
um and diameter of 400 um, which can be used to compute a droplet
volume of 5.4 nL. Table 2 shows exemplary nano-droplet volumes
(e.g., nanoliter volume or less) obtained from multiple samples
after 1-second water or hybridization buffer dipping. To verify
that the droplet geometry is indeed a spherical cap, one can check
agreements between the droplet height, contact radius, and contact
angle. Subject to the spherical cap geometry, the nano-droplet
volume (V.sub.d) (e.g., nanoliter volume or less) can be
characterized by the droplet height (h), contact radius (R.sub.c),
radius of the sphere forming the spherical cap (R.sub.d), and the
contact angle (.theta.). Generally, two variables out of the four
are needed to characterize other variables. In some embodiments,
the relationships between these four variables can be expressed
as:
V d = 1 6 .pi. h ( 3 R c 2 + h 2 ) , R d = R c 2 + h 2 2 h ,
.theta. = 2 tan - 1 h R c ( 3 ) ##EQU00004##
Thus, the contact angle may be based on the droplet height and
contact radius, and obtained to be 44.6.degree., which is in
agreement with our measurement of 43.5.degree..
TABLE-US-00002 TABLE 2 Nano-droplet volumes after 1-second water
dipping Sample # Nano-liter droplet Volume (nl) 1 5.4 2 5.8 3 5.3 4
5.1 5 5.5 6 5.6 Mean 5.4 Standard Deviation 0.3
[0173] The water dipping process forms nano-droplets (e.g.,
nanoliter volume or less) in a massively parallel fashion due to
low variances in the SiO.sub.2 pattern size and contact angle of
the water-SiO2 interface. In exemplary device setup, the SiO.sub.2
pattern size is patterned onto the silicon wafer by
photolithography, which offers low variance (+/-1 micron) for 400
microns patterning. The contact angle, on the other hand, may be
determined by interfacial energy and hysteresis. During water
immersion and removal, a thin layer of water is formed homogenously
across the array device. Because the water on top of black silicon
is in a dynamically unstable Cassie state, as water on the edge of
the device dissipates, the entire water layer on black silicon
follows and leaves nano-droplets (e.g., nanoliter volume or less)
on SiO.sub.2 patterns. The nano-droplets (e.g., nanoliter volume or
less) are formed by a self-assembled process in which the water
cohesion force is greater than the adhesion force between water and
black silicon, thus stripping waters from the surface. In contrast,
water cohesion force is less than the adhesion force between water
and SiO.sub.2, thus forming nano-liter water droplets. Because both
water cohesion and surface adhesion forces are material properties
that vary with the purity of water, SiO.sub.2 and black silicon
surface consistency, and temperature, the contact angle of the
nano-droplet (e.g., nanoliter volume or less) is intrinsically
homogenous across the microarray. The nano-droplets (e.g.,
nanoliter volume or less) have a droplet size standard deviation of
300 pL (Table 2). Table 3 shows the setup of three example nucleic
acids used for synthetic miR-205 DNA mimic quantification. Overall,
the homogeneity of both SiO.sub.2 pattern size and contact angle
results in a massively parallel, self-assemble nano-droplet (e.g.,
nanoliter volume or less) microarray.
TABLE-US-00003 TABLE 3 Single strand DNA for Synthetic miR-205 DNA
mimic quantification SEQ Sequence ID Modifi- Name NO Sequence
(5'-3') cation Length DNA Probe 1 TGC GAC CTC AGA CTC 3' 40 nt CGG
TGG AAT GAA GGA C6Amine AAA AAA AAA A Scrambled 5 AGC AGG AGA TAC
GAC 3' 40 nt DNA ATA ATA CAC GAT AAG C6Amine Probe TAG ACA CGA G
DNA Target 6 TCC TTC ATT CCA CCG 3' 30 nt (Synthetic GAG TCT GAG
GTC GCA Biotin miR-205 DNA mimic)
[0174] Mass transfer occurs when there is a concentration gradient
across two connected systems. Nevertheless, with brief dipping
process, the majority of the nucleic acids are retained on the
SiO.sub.2 pattern. During water or hybridization buffer immersion,
there is a significant nucleic acid concentration gradient across
the SiO.sub.2 pattern and the liquid reservoir, and thus after
water or hybridization buffer removal, loss of nucleic acid from
mass transfer is expected. The relationship between water immersion
duration and nucleic acid retention is quantified by drying
fluorescently labeled DNA on SiO.sub.2 patterns and recording the
fluorescent signal for water immersion time varying from 1-13
seconds. FIG. 24 shows DNA retained on SiO.sub.2 surface based on
the measured fluorescent intensity after drying from water
immersion at different immersion lengths of 0, 1, 2, 3, and 13
seconds, respectively. DNAs used in the exemplary device are 24
nucleotides in length and labeled with FAM. Relative fluorescent
units are calculated by normalizing the integrated intensity with
respect to 0 seconds water immersion. While a significant decay in
fluorescent signal is observed for 2-seconds water immersion,
.about.90% of the original signal is retained for 1-second water
immersion.
[0175] To demonstrate the performance of the nano-droplet (e.g.,
nanoliter volume or less) microarray device, we present data on DNA
target sensitivity curve, dynamic range, and accelerated
hybridization kinetics. Particularly, synthetic miR-205 DNA mimic
was chosen to be the DNA target to illustrate applications for
short, 30mer oligonucleotides quantification. In the disclosed
device workflow, DNA targets are dried onto the SiO.sub.2 pattern
and re-suspended in nano-droplets (e.g., nanoliter volume or less).
Depending on the initial solution volume prior to drying, different
degrees of enrichment can be achieved by the disclosed workflow and
thus resulting in different sensitivity curves.
[0176] We have implemented two enrichment conditions with initial
solution volumes of 1 mL and 5 .mu.L. FIGS. 25A-25D show DNA target
(e.g., Synthetic miR-205 DNA mimic) sensitivity from 1 ml to 5
.mu.l and then from 5 .mu.l to 5 nl enrichment. FIG. 25A shows 1 ml
to 5 .mu.l enrichment achieved by evaporating target DNA solution
at 95.degree. C. while Teflon mold restricts solution above the
SiO.sub.2 patterns. FIG. 25B shows 5 .mu.l to 5 nl enrichment
achieved by evaporating target DNA solution at 50.degree. C.,
followed by nano-liter droplet self-assembly described in FIG. 22E.
When starting with 1 mL of initial volume, the solution undergoes a
two-step drying process that begins with an accelerated evaporation
at 95.degree. C. from 1 ml to 5 .mu.L, and followed by a slower
evaporates from 5 .mu.L to dry at 50.degree. C. However, when
starting with 5 .mu.L of initial volume, the solution evaporates to
dry in one step at 50.degree. C. While evaporation time minimizes
when the drying process is carried out near the boiling point, in
practice we reduces evaporation temperature when DNA target
solution volume decreases to 5 .mu.L. This temperature control is
needed to prevent droplet state transition that can hinder solution
self-realignment to the SiO.sub.2 pattern.
[0177] Briefly, water in contact with nano-patterned,
super-hydrophobic surface such as black silicon can existed in
either Cassie or Wenzel states. Cassie state is a state in which
the liquid is in contact with only the nano-pattern peaks and thus
trapping air pockets underneath and demonstrating unstable
dynamics. Wenzel state, on the other hand, describes full liquid
contact with the surface and shows significant resistance to
movement. Simulation predicts equal energy level for both states
separated by a free-energy barrier and states coexistence. Yet,
experimental investigations revealed a lowered energy level in
Wenzel state, indicating an irreversible state transition.
Furthermore, the state-transition is initiated via a nucleation
event that is dependent of the contact area and thermal energy. At
DNA target solution volume of 5 .mu.L, evaporation is carried out
at 50.degree. C. to minimize nucleation event that initiates the
Cassie-Wenzel state transition. Premature Cassie-Wenzel state
transition during evaporation causes redeposition of dried DNA
target outside the intended SiO2 pattern. By switching from
95.degree. C. to 50.degree. C. at different stage of drying, the
dried target area coverage closely resembles that of the SiO.sub.2
pattern size.
[0178] Both drying processes starting at different initial volumes
conclude with water or hybridization buffer dipping that generates
homogenous, 5.4 nL of nano-droplets. The resulting volume changes
lead to solute concentration enrichment of 1,000 and 200,000 times
respectively for initial volume of 5 .mu.L and 1 mL. These 2
enrichment ratios translate directly into the sensitivity curve
observed: 1 mL and 5 .mu.L initial volumes correspond to
sensitivity curves covering 50 aM to 500 fM and 10 fM to 100 pM DNA
target concentration (see FIGS. 25C-25D). FIG. 25C shows that 1 ml
to 5 nl enrichment results in a linear sensitivity curve ranging
from 50 aM to 500 fM. FIG. 25D shows that 5 .mu.l to 5 nl
enrichment results in a linear sensitivity curve ranging from 10 fM
to 100 pM.
[0179] The signal is expressed in number of quantum dots counted on
the 145.times.108 micron detection area, and negative control
quantum dots count resulted from scrambled sensing DNA is
subtracted from the signal.
[0180] Moreover, given the same nano-droplet volume (e.g.,
nanoliter volume or less), knowing the copy number of DNA target is
sufficient to determine the final quantum dot signal, regardless of
particular initial volume or target concentration. For instance, 1
mL of 5 fM DNA target and 5 .mu.L of 1 pM DNA target display almost
identical quantum dot binding signal in our sensitivity curve
because both contain .about.5 amole of DNA target. Still, we
observed slightly lowered quantum dot binding systemically with
initial volume of 1 mL due to either the result of the extensive
drying process or possible molecule adherence to the Teflon mold
surface.
[0181] Besides absolute copy number, DNA target concentration also
provides important biologically relevant information, and thus a
wide concentration dynamic range is desired. On a log-log plot,
both 1 mL and 5 .mu.L initial volume sensitivity curves in FIGS.
25C-25D display linear relationship between the quantum dot binding
signal and the DNA target concentration, exhibiting 4 orders of
dynamic range. Sensitivity curve's dynamic range is intrinsically
limited by optical diffraction and photonic sensor performance. By
combining the two sensitivity curves with partially overlapping
sample concentration range for cross-reference, a dynamic range of
over 6 orders of magnitude is demonstrated. The slope for these
linear curves is less than 1 for both cases (0.7 for 1 mL and 0.8
for 5 .mu.L initial volume), meaning that the overall binding
efficiency reduces as target DNA concentration increases. The
effect of DNA probe surface density on binding efficiency is well
studied: increasing DNA probe surface density may lead to reduced
binding efficiency, which is caused by steric hindrance and
electrostatic repulsion. Because quantum dots have high negative
surface charges, electrostatic repulsion is likely the cause of the
reducing overall binding efficiency with increasing target DNA
concentration.
[0182] In conventional microarrays, extended hybridization time
(16+ hours) is required for DNA targets to diffuse from bulk
solution toward surface DNA probes. Due to enrichment by
evaporation and reduced volume of the nano-droplet (e.g., nanoliter
volume or less) array, hybridization time in the disclosed
technology is reduced to 5 minutes. This can be at least partially
attributed to the nano-droplet (e.g., nanoliter volume or less)
height, or the target DNA maximum diffusion length, which is
.about.80 microns, as oppose to .about.2 mm in the case of a 4
.mu.l droplet. In a model of one-dimensional target DNA-probe DNA
binding, the binding efficiency (.eta.(t)) is diffusion limited and
related to diffusion coefficient of the target (DT), time (t), and
diffusion length (L) as following:
.eta. ( t ) .varies. 1 - - D T .pi. 2 t ( 2 L ) 2 . ( 4 )
##EQU00005##
According to this model, a diffusion length reduction from 2 mm to
80 micron (25 fold) leads to time reduction of 625 folds;
therefore, the time required for completing hybridization can
theoretically be accelerated from 16 hours to 90 seconds.
[0183] FIG. 26 shows hybridization completion rate vs.
hybridization time within the nano-droplets (e.g., nanoliter volume
or less). 10 pM of Synthetic miR-205 DNA mimic enriched from 5
.mu.l to 5 nl is allowed to hybridize from 5 minutes to 2 hours in
nano-droplets (e.g., nanoliter volume or less). The signal is
expressed in number of quantum dots counted on the 145.times.108
micron detection area, and negative control quantum dots count
resulted from scrambled sensing DNA is subtracted from the signal.
As shown in FIG. 26, the accelerated hybridization is demonstrated
by incubating the nano-droplet (e.g., nanoliter volume or less)
array for various amount of time. After 5 minutes of hybridization,
hybridization is completed binding efficiency cease to increase
with time. Nevertheless, a significant binding efficiency is
observed without any incubation, suggesting hybridization
occurrence during the drying process.
[0184] Lastly, a comprehensive evaluation of the nano-droplet
(e.g., nanoliter volume or less) microarray is addressed across
many criteria including maximum sensitivity, linear dynamic range,
hybridization time, and throughput. Table 4 shows the comparison of
maximum sensitivity, linear dynamic range, hybridization time, and
throughput of the disclosed nano-droplet (e.g., nanoliter volume or
less) microarray and other recent microarray-based techniques for
nucleic acid quantification. Compared to other recent
microarray-based techniques, nano-droplet (e.g., nanoliter volume
or less) microarray disclosed in this patent document shows
significant improvements in terms of sensitivity, linear dynamic
range, and hybridization time (Table 4). While most other methods
focus on improvements in nucleic acid chemistry, surface
treatments, and microfluidic design, nano-droplet (e.g., nanoliter
volume or less) array relies on physical enrichment of DNA targets
and accelerated hybridization kinetics due to its miniature droplet
dimension. To summarize, the nano-droplet (e.g., nanoliter volume
or less) microarray has achieved a maximum sensitivity of 50 aM (50
zmol), 6 orders or linear dynamic range, 5 minutes of hybridization
time, and a throughput similar to current microarray platforms.
TABLE-US-00004 TABLE 4 Comparison of maximum sensitivity, linear
dynamic range, hybridization time, and throughput of the
"Nano-droplet (e.g., nanoliter volume or less) Microarray" and
other recent microarray-based methods for nucleic acid
quantification. Linear Detection Maximum Dynamic Hybridization
Throughput Method method Sensitivity Range Time (+) Nano-droplet
(e.g., Quantum Dots 50 aM/ 6 orders 5 min +++ nanoliter volume or
50 zmol less) Microarray (disclosed technology) Microconcentration
Fluorescence 100 pM/ 3 orders 30 min +++ (Texas Red) 2 fmol
Oil-encapsulated Quantum Dots 100 fM/ 2 orders 30 min +++
nano-droplet array 400 zmol Direct and sensitive Fluorescence 5 fM/
4 orders 20-48 hr +++ miRNA profiling (Cy3 and Cy5) 200 zmol
Ternary Surface Electrochemistry 10 fM/ 5 orders 30 min ++
Monolayers (HRP enzyme) 40 zmol Ultra-sensitive Quantum Dots 10 fM/
3 orders 1 hr + single-molecule 500 zmol detection Two-temperature
Fluorescence 10 fM/ 4 orders 16 hr.sup. +++ hybridization with
(Cy3) 1 amol LNA probes Paper-based solid- Quantum Dots- 300 fmol 1
orders 30 min ++ phase assay Cy3 FRET Duplex-specific
Electrochemistry 1 fM 2 orders 12 hr.sup. ++ nuclease
Isotachophoresis Fluroescence 100 fM 3 orders 30 min +++ (Cy3)
Materials and Processes
Nano-Liter Droplet Array Process Flow
[0185] Three example nucleic acids tested are listed in Table 3.
After surface pretreatment, SiO2 pattern surfaces are cross-linked
with DNA probes (see FIG. 22B). A volume of 5 ul or 1 ml solution
of target DNA is added onto SiO2 pattern (see FIG. 22C) and allowed
to evaporate at controlled temperature. While evaporating, the DNA
target solution droplets are realigned onto SiO2 patterns, shrink,
and are dried completely (see FIG. 22D) onto SiO2 patterns.
Immersing the device with dried DNAs in water or hybridization
buffer for 1 second and removing the device quickly (see FIG. 22E),
nano-droplets (e.g., nanoliter volume or less) with resuspended
DNAs are self-assembled on SiO2 patterns. To avoid evaporation of
droplets, oil is immediately added onto the nano-liter droplets,
ceasing evaporation (see FIG. 22F). After 5 minutes incubation at
50.degree. C., DNA probes and targets hybridize and form DNA
duplexes. Finally, SiO.sub.2 patterns are coated with milk protein
to prevent non-specific binding and the duplexes are labeled with
quantum dots for observation (see FIG. 22G).
Device Fabrication
[0186] Embodiments of the device fabrication technique have been
described earlier in this patent document. Briefly, 400-micron
diameter circular patterns are formed on a mechanical grade silicon
wafer by conventional photolithography, and then coated with SiO2
and a protective chromium layer. By deep reactive ion etch (DRIE),
super-hydrophobic black silicon surface forms around the patterns.
Lastly, the chromium protective layer is removed by chromium
etchant.
Device Surface Pretreatment
[0187] Embodiments of the device surface pretreatment prior to the
nano-droplet (e.g., nanoliter volume or less) hybridization array
workflow have been described earlier in this patent document.
Following device fabrication, SiO.sub.2 island surfaces are coated
with aminosilane (APTES) and then functionalized with aldehyde. DNA
probe and scrambled DNA probe (negative control) are spotted on the
SiO.sub.2 islands via crosslinking reaction between the amine 3'
modification of the DNA probe and the aldehyde functional group on
SiO.sub.2 island surfaces.
Image Analysis and Quantum Dot Counting
[0188] Sample images are taken under 100.times. oil immersion lens
(e.g., Nikon, NA1.45) with an enclosed fluorescent microscope
(e.g., Keyence BZ-9000). The samples are excited by mercury
lamplight filtered through a single-band bandpass filter (e.g.,
Samrock, 405/10 nm) and the emission light is filtered by another
single-band bandpass filter (e.g., Samrock, 536/40 nm). Raw images
taken from the microscope are processed through haze reduction and
black balance algorithms. Finally, the quantum dots remaining on
the SiO.sub.2 islands are counted using object size, connectivity,
and intensity filters integrated in an object counter module
included in the microscope software (e.g., BZ-II Analyzer). The raw
images are captured within a detection area of 145.times.108
.mu.m.sup.2. In principle, each quantum dot signifies a single DNA
target hybridized with the probe, yet quantum dot residues might be
left on the surface due to incomplete wash. In some embodiments,
the real signal in the sensitivity curves and hybridization
time-series can be expressed as the difference between the quantum
dots counts for the DNA probe and scrambled DNA probe with the same
target DNA (e.g., synthetic miR-205 DNA mimics) concentration.
Discussion
[0189] Various embodiments of the disclosed technology has
established and validated an ultra-sensitive nucleic acid
quantification technique and device based on the nano-droplet
(e.g., nanoliter volume or less), on-chip concentration array
platform. The detection scheme utilized the patterned black-silica
surface to induce self-assembly mechanism of nano-droplets (e.g.,
nanoliter volume or less) formation, allowing precise control of
sample volumes and massively parallel processing capability.
Coupled with the rapid evaporation feature of the device, the
samples can be greatly enriched to enable nucleic acid detection at
both 50 aM to 500 fM and 10 fM to 100 pM concentrations resulted in
a combined wide linear dynamic range of 6-orders. Moreover, the
reduced sample droplet volume facilitates hybridization reaction to
within 5 minutes and greatly shortens the time scale required for
the quantification process. Combining the features above, the
disclosed technology demonstrates a unique nucleic acid detection
system with significantly improved sensitivity and speed over
existing microarray platforms.
[0190] While this patent document contain many specifics, these
should not be construed as limitations on the scope of any
invention or of what may be claimed, but rather as descriptions of
features that may be specific to particular embodiments of
particular inventions. Certain features that are described in this
patent document in the context of separate embodiments can also be
implemented in combination in a single embodiment. Conversely,
various features that are described in the context of a single
embodiment can also be implemented in multiple embodiments
separately or in any suitable subcombination. Moreover, although
features may be described above as acting in certain combinations
and even initially claimed as such, one or more features from a
claimed combination can in some cases be excised from the
combination, and the claimed combination may be directed to a
subcombination or variation of a subcombination.
[0191] Similarly, while operations are depicted in the drawings in
a particular order, this should not be understood as requiring that
such operations be performed in the particular order shown or in
sequential order, or that all illustrated operations be performed,
to achieve desirable results. Moreover, the separation of various
system components in the embodiments described in this patent
document should not be understood as requiring such separation in
all embodiments.
[0192] Only a few implementations and examples are described and
other implementations, enhancements and variations can be made
based on what is described and illustrated in this patent
document.
* * * * *