U.S. patent application number 12/568651 was filed with the patent office on 2010-06-17 for selective capture and release of analytes.
Invention is credited to Jingyue Ju, Donald W. Landry, Qiao Lin, ThaiHuu Nguyen, Renjun Pei, Chunmei Qiu, Milan N. Stojanovic.
Application Number | 20100151465 12/568651 |
Document ID | / |
Family ID | 42240992 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100151465 |
Kind Code |
A1 |
Ju; Jingyue ; et
al. |
June 17, 2010 |
Selective Capture and Release of Analytes
Abstract
The described subject matter includes techniques and components
for minimally invasive, selective capture and release of analytes.
An aptamer is selected for its binding affinity with a particular
analyte(s). The aptamer is functionalized on a solid phase, for
example, microbeads, polymer monolith, microfabricated solid phase,
etc. The analyte is allowed to bind to the aptamer, for example, in
a microchamber. Once the analyte has been bound, a temperature
control sets the temperature to an appropriate temperature at which
the captured analyte is released.
Inventors: |
Ju; Jingyue; (Englewood
Cliffs, NJ) ; Landry; Donald W.; (New York, NY)
; Lin; Qiao; (New York, NY) ; Nguyen; ThaiHuu;
(Richmond, VA) ; Pei; Renjun; (New York, NY)
; Qiu; Chunmei; (New York, NY) ; Stojanovic; Milan
N.; (Forth Lee, NJ) |
Correspondence
Address: |
BAKER BOTTS L.L.P.
30 ROCKEFELLER PLAZA, 44TH FLOOR
NEW YORK
NY
10112-4498
US
|
Family ID: |
42240992 |
Appl. No.: |
12/568651 |
Filed: |
September 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US08/58433 |
Mar 27, 2008 |
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12568651 |
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61165690 |
Apr 1, 2009 |
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61171333 |
Apr 21, 2009 |
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Current U.S.
Class: |
435/6.12 ;
422/69; 435/287.2; 436/164 |
Current CPC
Class: |
C12Q 1/6816 20130101;
G01N 1/4055 20130101; G01N 33/5308 20130101; C12Q 1/6816 20130101;
Y10T 436/25375 20150115; Y10T 436/24 20150115; G01N 21/6408
20130101; Y10T 436/143333 20150115; C12Q 2525/205 20130101; C12Q
2565/629 20130101; C12Q 2527/107 20130101 |
Class at
Publication: |
435/6 ; 436/164;
435/287.2; 422/69 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 21/63 20060101 G01N021/63; C12M 1/34 20060101
C12M001/34; G01N 33/00 20060101 G01N033/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with government support under Grant
Nos. CBET-0693274 and EIA-324845 awarded by The National Science
Foundation. The government has certain rights in the invention.
Claims
1. A system for capture and selective release of an analyte,
comprising: a solid phase; an aptamer functionalized on the solid
phase, the aptamer adapted to bind the analyte; and a temperature
regulator thermally coupled to the aptamer for setting the
temperature of the aptamer to a set point, the analyte released
from the aptamer at the set point.
2. The system of claim 1, wherein the analyte initially exists in
an impure form and the impurities are removed with a washing
solution after the analyte is bound to the aptamer.
3. The system of claim 1, wherein the analyte includes an
oligonucleotide.
4. The system of claim 1, wherein the solid phase includes a
microbead.
5. The system of claim 1, wherein the analyte is in an aqueous
solution.
6. The system of claim 1, further comprising: a collector for
collecting the released analyte; and a detector for measuring the
amount of analyte released.
7. The system of claim 6, wherein the collector includes a spotting
well.
8. The system of claim 6, wherein the detector is a mass
spectrometer.
9. The system of claim 1, further comprising: a microchannel for
receiving the released analyte and directing the released analyte
through a hydrophobic valve.
10. The system of claim 1, wherein the system is incorporated on a
microfluidic chip platform.
11. A method for capturing and selectively releasing an analyte,
comprising: (a) binding the analyte to an aptamer, the aptamer
functionalized on a solid phase; and (b) setting the temperature of
the aptamer such that the analyte is released from the aptamer.
12. The method of claim 11, further comprising: (c) introducing the
analyte to the aptamer in an impure form; (d) washing the bound
aptamer analyte complex to remove impurities; and (e) repeating
(c), (a), and (d) such that the amount of bound analyte is
increased.
13. The method of claim 11, further comprising: (f) collecting and
detecting the analyte.
14. The method of claim 11, wherein the detecting includes
performing mass spectrometry on the released analyte.
15. The method of claim 11, wherein the detecting includes
detecting fluorescence intensity.
16. The method of claim 11, wherein the solid phase includes a
microbead.
17. The method of claim 11, wherein the analyte includes an
oligonucleotide.
18. A method for selectively increasing the concentration of an
analyte, comprising: (a) functionalizing a solid phase with an
aptamer; (b) introducing the analyte to the aptamer in an impure
form; (c) binding the analyte to the aptamer; (d) washing the bound
aptamer analyte complex to remove impurities; (e) repeating (a)-(d)
until a desired analyte concentration is reached; and (f) setting
the temperature of the aptamer such that the analyte is released
from the aptamer.
Description
PRIORITY CLAIM
[0001] This application is a continuation-in-part of and claims
priority to International Application Serial No. PCT/U.S.08/058,433
and also claims priority to U.S. Provisional Application Ser. Nos.
61/165,690, filed Apr. 1, 2009, and 61/171,333, filed Apr. 21,
2009, which are incorporated by reference in their entireties.
BACKGROUND
[0003] The present application relates to, but is not limited to,
selective capture and release of analytes. For example, the present
application relates to minimally invasive extraction, purification
and concentration (PC) of analytes.
[0004] A need exists for techniques to selectively capture and
release analytes with minimal harm to the analytes. For example,
such techniques are applicable to extraction of analytes for
biochemical analysis. Other applications include detection of
harmful components in pharmaceuticals or food, extraction of
harmful environmental agents, selective release of drugs at a
target location in the body, and the like.
[0005] As an example, there is a desire to develop highly
integrated biological analysis devices that can be used to perform
general biochemical analysis. One component in these devices is
sample preparation, which involves extraction and PC of applicable
analytes.
[0006] Some techniques have employed solid-phase (SP) gels for
retention of target molecules. A common shortcoming of SP devices
is that their capture mechanisms are often indiscriminate with
respect to the target analyte. For example, hydrophobic and
ion-exchange SP device are limited because they extract impure
compounds with similar physical or chemical properties as the
target. With applications in drug delivery or chemical assays,
where specific molecules need to be released, introducing
impurities can be problematic. In addition, elution of molecules
using harsh pH or solvent gradients is common in SP devices. For
certain biomedical applications, these elution schemes can present
potential health hazards. Furthermore, it is desirable to
selectively release the captured molecules for applications in
which their use is location-specific.
[0007] Biotechnology research, such as proteomics and genomics,
utilizes biological mass spectrometry, which is label-free and
offers increased resolution detection. In particular, matrix
assisted laser desorption/ionization mass spectrometry (MALDI-MS)
is useful because it permits relatively simple data interpretation,
good detection limits and parallel processing. MALDI-MS is based on
a soft ionization technique in which analytes are cocrystallized
with an energy-absorbing matrix material on the surface of a
substrate (called a MALDI analysis plate). Notwithstanding its
broad utility, the overall quality and efficacy of quantifiable
MALDI-MS generally depends on the purity of the introduced sample.
Techniques involving sample preparation, such as analyte
extraction, have increasingly been employed to condition biological
samples prior to MALDI-MS analysis. This can entail the separation,
purification and concentration of analytes preceding quantitative
analysis. For example, analyte extraction can be used to retrieve
and isolate a rare analyte from a complex mixture of undesirable
constituents such as salts, particulates, solvents or physiological
tissue so as to enrich and enhance the analyte's MALDI-MS
detection.
[0008] Solid-phase extraction (SPE) as a sample preparation
procedure prior to MALDI-MS that can be used to provide pure and
concentrated samples to enable increased sensitivity analysis.
During SPE, an analyte of interest within a fluid phase is exposed
to a solid phase (e.g., microbeads coated with a thin layer of a
functional material). The analyte interacts through surface
chemistry with the coating and therefore is retained by the solid
phase. This allows impurities and non-target compounds remaining in
the liquid phase to be removed by rinsing. Next, a reagent (such as
an organic solvent) is generally used to disrupt the interaction
between the solid phase and the analyte, thereby eluting the
analyte for further analysis. Other sample preparation techniques
include electrokinetic sample stacking, liquid-liquid extraction,
and dialysis. The off-line nature of MALDI-MS makes it suitable for
coupling to off-line SPE approaches, which facilitate
high-throughput processing designs with small dead volumes.
[0009] In some SPE protocols, one challenge is to effectively
concentrate and purify minute quantities of analytes, while
minimizing absorptive losses and maximizing recovery in as compact
an elution volume as possible. Microfluidic technology has been
utilized to attempt to overcome this obstacle. Miniaturization
helps facilitate the handling of limited sample quantities, the
reduction of dead volumes, an increase the effective
surface-to-volume ratio to promote efficient chemical reactions,
and integration. Also, microfabrication allows for massive
parallelization of sample processing, while being amenable to
MALDI-MS which can lower analysis costs. Existing microfluidic SPE
devices utilize physisorption capture of the target analyte by gels
or membranes. For example, some techniques use a commercial
reversed-phase gel (Poros) on some microfabricated silicon chips
for sample enrichment of alcohol dehydrogenase. The proteins are
eluted by addition of a polar solvent (e.g., acetonitrile), which
changes the surface polarity of the support to release the bound
analyte. Ion-exchange supports, such as some methacrylate based
gels, depend on adjustment of charged molecules on the retention
media to interact with analytes. Strong pH reagents can be
introduced to subsequently release the molecules of interest.
Alternatively, other techniques use a packed 2.5 mm column of C18
microbeads for the reverse-phased preconcentration of ephedrine on
a poly (vinylpyrrolidone) chip which is then eluted using an
acetonitrile-borate buffer solution.
[0010] Existing microfluidic SPE devices, however, remain
inadequate to address the current demands in MALDI-MS analysis,
which increasingly requires processing of complex biological or
chemical samples, such as blood, serum, or tissue mass. A given
analyte should be detectable amongst cellular debris, non-specific
molecules, and salts within such samples. Standard functional
chemistries for solid-phase purification often lack selectivity to
target analytes since impurities usually exhibit similar physical
properties (e.g., hydrophobicity or ionic charge) which allow their
simultaneous retention. For unambiguous, sensitive detection of
biomolecules by MALDI-MS, it is useful that the analyte extraction
be specific, e.g., the analyte and no impurities are retained by
the solid phase. Moreover, recovery of biomolecules using
traditional techniques generally requires an adjustment in pH or
application of a solvent gradient. This can compromise the
integrity of sensitive compounds (which can already be in rare
supply) and can further complicate the protocol by requiring the
handling of potentially harsh reagents.
[0011] Biosensors are used for the detection and analysis of
biomolecules that are disease relevant biomarkers such as genes,
proteins, and peptides. They can include of a molecular recognition
component and a transducer converting the binding event into a
measurable physical signal. An important class of biosensors
includes affinity biosensors, which rely on highly selective
affinity receptors recognizing target biomolecules. Traditionally
used affinity receptors include antibodies and enzymes, which are
known to have limitations such as instability, poor regeneration,
and physiologically-dependent production. These limitations can be
addressed by biosensors that employ alternative, synthetically
generated affinity receptors, in particular aptamers.
[0012] Aptamers include oligonucleotides that recognize target
molecules specifically by highly selective affinity interaction;
they are isolated through a synthetic procedure called systematic
evolution of ligands by exponential enrichment (SELEX), whereby
very large populations of random sequence oligomers (DNA or RNA
libraries) are screened against the target molecule in an iterative
procedure. Aptamers have been developed to target a variety of
biomolecules (e.g., small molecules, peptides, and proteins) in
diverse applications, such as target validation, drug discovery,
and in particular, diagnostics and therapy. The intense attention
received by aptamers can be attributed not only to their high
specificity, but also to characteristics that are lacking in more
established affinity receptors such as enzymes, lectins, and
antibodies. These include enhanced stability at room temperature,
and more easily modified terminal ends, as compared to their
conventional affinity receptor counterparts (e.g., antibodies and
enzymes), so as to facilitate attachment to stationary surfaces.
Moreover, aptamer-target binding is generally reversible under
changes in environmental parameters such as pH and temperature.
Thus, aptasensors can be regenerated via such experimental stimuli,
which can also be exploited to allow controlled release and
recovery of target biomolecules.
[0013] Microelectromechanical systems (MEMS) have been applied to
biosensing, leading to minimized sample consumption, improved
robustness and reliability, reduced costs, and the possibility of
parallelized, high-throughput operation. In particular,
microfluidic devices have been used for affinity biosensing, such
as microcantilever immunosensors for myoglobin and
nanoparticle-antibody conjugated array sensors for detecting
food-born Escherichia coli. Microcantiliever aptasensors have been
used for specific detection of Thermus aquaticus DNA polymerase.
Biomolecules are detected after binding by monitoring surface
stress induced deflection of the cantilever by an interrogating
light source. Alternatively, love-wave microfluidic aptasensors
have been used to detect multifunctional serine protease thrombin
and Rev peptide, fabricated from polymethylmethacrylate on top of a
quartz substrate. Nanostructures such as single-walled carbon
nanotubes have been functionalized with aptamers to detect
thrombin. The selectivity of the thrombin aptamer has been tested
against elastase to which the conductance of the SWNT-FET showed no
change.
[0014] Aptasensing of arginine vasopressin (AVP) for the diagnosis
and therapy of septic shock (induced by severe infection) and
congestive heart failure, conditions that restrict the
cardiovascular system's ability to provide adequate perfusion in
order to maintain organ functionality is a clinical application of
aptasensors. Both disorders are indicated by elevated levels of
AVP, a cyclic polypeptide neurohormone that is synthesized in the
hypothalamus and promotes vasoconstriction. Specifically,
physiological concentrations of AVP in plasma markedly increases up
to tenfold that of average levels (5-10 pM) in order to maintain
arterial pressure and hence, blood perfusion. As shock progresses
however, the initial abundance of AVP in plasma decreases. Thus,
the ability to monitor and control AVP over time can reveal the
homeostatic status of the patient, and could potentially provide
therapeutic solutions for septic shock and congestive heart
failure. Platforms for vasopressin include immunoradiometric assays
(IRA) and enzyme-linked immunosorbent assays (ELISA). The use of
these assays is often hindered by several limitations:
time-consuming and complicated radio and fluorescent labeling
protocols; excessive use of sample and auxiliary reagents; and
limited long-term stability and shelf-life. Moreover, prolonged
incubation times can result in slow diagnostic turnaround (3-11
days), which renders these techniques rather ineffective for
therapeutic management of AVP.
SUMMARY
[0015] Systems and methods for selective capture and release of
analytes are disclosed herein.
[0016] Some embodiments include components for capture and
selective release of an analyte. In an exemplary apparatus, a
system includes a solid phase, an aptamer functionalized on the
solid phase for binding the analyte, and a temperature regulator
for setting a temperature to a set point, such that the analyte is
released from the aptamer at the set point. The analyte can
initially exist in an impure form and the impurities can be removed
with a washing solution after the analyte is bound to the aptamer.
The analyte can include a peptide, protein, small molecule or live
cell. The solid phase can include a microbead. The analyte can be
in an aqueous solution.
[0017] The system can further include a collector for collecting
the released analyte; and a detector for measuring the amount of
analyte released. The collector can include a spotting well. The
detector can be a mass spectrometer. The components can further
include a microchannel for receiving the released analyte and
directing the released analyte through a hydrophobic valve. These
components can be incorporated on a microfluidic chip platform.
[0018] Techniques for capturing and selectively releasing an
analyte are also provided. In some embodiments, the techniques
include binding the analyte to an aptamer, the aptamer
functionalized on a solid phase, and setting the temperature of the
aptamer such that the analyte is released from the aptamer. The
procedure can further include introducing the analyte to the
aptamer in an impure form and washing the bound aptamer analyte
complex to remove impurities. The procedural elements can be
repeated so that the amount of bound analyte is increased. The
procedure can further include collecting and detecting the analyte.
The detecting can include performing mass spectrometry on the
released analyte or detecting fluorescence intensity.
[0019] Techniques for selectively increasing the concentration of
an analyte are also provided. In some embodiments, the techniques
include functionalizing a solid phase with an aptamer, introducing
the analyte to the aptamer in an impure form, binding the analyte
to the aptamer, and washing the bound aptamer analyte complex to
remove impurities. The procedural elements can be repeated so until
a desired analyte concentration is reached, and the temperature of
the aptamer can be set such that the analyte is released from the
aptamer.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0021] The accompanying drawings, which are incorporated into and
constitute part of this disclosure, illustrate preferred
embodiments of the described subject matter and serve to explain
the principles of the described subject matter.
[0022] FIG. 1 is a schematic drawing showing an example device
according to one embodiment of the described subject matter.
[0023] FIG. 2 depicts an example according to an embodiment of the
described subject matter. The figure shows a fabrication technique:
2a-c show polydimethysiloxane (PDMS) channel techniques, 2d-e show
processing of integrated heater and temperature sensor, and 2f
shows a complete package.
[0024] FIG. 3 depicts an example according to an embodiment of the
described subject matter. The figure shows the relationship of
fluorescence signal to TO-AMP concentration in solution.
[0025] FIGS. 4(a)-(b) depict exemplary aptameric concentration of
analytes using an example device according to the described subject
matter. FIG. 4(a) shows concentration of an analyte using
consecutive injections of a 200 nM dilute solution, while FIG. 4(b)
shows concentration of an analyte using consecutive injections of a
500 nM injections.
[0026] FIG. 5 depicts thermal release of AMP from bead surfaces
incubated with 10 .mu.M injection according to an embodiment of the
described subject matter.
[0027] FIG. 6 depict extraction of AMP after release at (a)
75.degree. C., (b) 85.degree. C., and (c) 95.degree. C. according
to exemplary embodiments of the described subject matter.
[0028] FIGS. 7(a)-(b) depict (a) chemical structure of bio-ATP-40-1
aptamer and (b) molecular structure of TO-AMP according to
exemplary embodiments of the described subject matter.
[0029] FIG. 8 depicts an example layout of the microfluidic SPE
device used for this demonstration according to an exemplary
embodiment of the described subject matter
[0030] FIGS. 9(a)-(f) depict a simplified device flow: (a-d) a
microchannel realized with standard soft lithography techniques and
(e & f) a package according to exemplary embodiments of the
described subject matter. Line A-A' in the schematic of FIG. 8 is a
cross-section reference
[0031] FIG. 10 depicts a bright-field micrograph of the chamber,
including a magnified area for fluorescence imaging and processing
according to an exemplary embodiment of the described subject
matter
[0032] FIGS. 11(a)-(b) depict time resolved extraction of TO-AMP
(400 nM) by ATP-aptamer according to an exemplary embodiment of the
described subject matter. Measurements are made in the (A-A')
direction of the linked micrograph as shown in FIG. 11(a). At each
time level, fluorescence intensity is sampled, averaged, and
normalized to produce a single value point for each interval.
[0033] FIGS. 12(a)-(e) depict micrographs displaying SPE extraction
of 3 concentrations of TO-AMP according to exemplary embodiments of
the described subject matter: (a) baseline fluorescence; (b) 400
nM; (c) 500 nM; and (d) 10 .mu.M.
[0034] FIGS. 13(a)-(b) depict controlled release of TO-ATP and
regeneration of an exemplary SPE device of the described subject
matter (baseline colinear w/horizontal axis). FIG. 13(a) shows
competitive displacement with ATP (800 .mu.M & 3.2 mM). FIG.
13(b) presents thermally induced release and regeneration.
Single-valued points are obtained similarly to time-resolved
data.
[0035] FIGS. 14(a)-(b) depict schematics of an example device
according to an embodiment of the described subject matter. FIG.
14(a) is an isometric view. FIG. 14(b) is an A-A' cross-sectional
view. The chip dimensions are 3.5.times.2.5.times.0.5 cm
(l.times.w.times.h).
[0036] FIGS. 15(a)-(e) depict a simplified device fabrication flow
example according to an embodiment of the described subject matter.
FIGS. 15(a-c) depict a microchannel and integrated heater and
temperature sensor elements realized with standard soft lithography
and MEMS fabrication techniques; FIGS. 15 (d & e) depict device
packaging according to exemplary embodiments of the described
subject matter.
[0037] FIGS. 16(a)-(b) depict operation of a passive valve: (a)
fluorescein solution flowing through the waste outlet bypassing
valve and (b) valving fluorescein solution through the valve
according to an exemplary embodiment of the described subject
matter.
[0038] FIG. 17 depicts spot size on a MALDI plate as a function of
flow rate used to transfer the sample to the deposition well
according to an exemplary embodiment of the described subject
matter.
[0039] FIGS. 18(a)-(b) depict MS from (a) a 0.1 .mu.M injected
sample and (b) a 1.0 .mu.M injected sample according to exemplary
embodiments of the described subject matter.
[0040] FIG. 19 depicts MS from an injected sample of AMP, CTP, UTP,
& GTP (1 .mu.M each) where AMP is isolated according to an
exemplary embodiment of the described subject matter.
[0041] FIGS. 20(a)-(b) depict MS from a sample spot obtained from
(a) 25 injections; (b) 250 injections of 10 nM AMP solution
according to exemplary embodiments of the described subject
matter.
[0042] FIG. 21 (a) depicts a schematic of the microchip
purification device with an inset illustrating the surface-tension
based valving scheme according to an embodiment of the described
subject matter; (b) depicts a cross-sectional view along line A-A
from (a) showing coupling scheme of the microchip to the MALDI
analysis plate for sample spotting before mass spectrometric
analysis according to an embodiment of the described subject
matter; (c) depicts a close-up photograph of a packaged device
according to an embodiment of the described subject matter.
[0043] FIG. 22 depicts an exemplary device fabrication flow
according to an embodiment of the described subject matter as seen
from cross-section A-A in FIG. 21a: (a-c) depicts SU-8 patterning
followed by subsequent PDMS prepolymer casting to form microfluidic
layers; (d) depicts glass substrate drilled for fluidic
interconnects; (e) depicts thermal evaporation and lift-off
patterning of Cr/Au bi-layer; (f) depicts PECVD deposition of
SiO.sub.2 passivation layer; (g) depicts microfluidic structural
layers aligned and permanently bonded to the glass substrate; (h)
depicts packaged chip with tubing.
[0044] FIG. 23 depicts MALDI-MS detection of varying concentrations
of AMP in a pure water solution using an ATP-aptamer functionalized
microchip coupled to a MALDI analysis plate according to an
embodiment of the described subject matter. (a) depicts
demonstrations using 10 nM, (b) 100 nM and (c) 1 .mu.M.
[0045] FIG. 24 depicts Discrete concentration after 25 (a) and 250
(b) infusions of a 10 nM AMP sample revealing detection enhancement
by aptamer-based enrichment according to an embodiment of the
described subject matter.
[0046] FIG. 25 depicts demonstration of the sample cleanup of a
model analyte apparatus before MALDI-MS detection according to an
embodiment of the described subject matter (a) MALDI spectrum of
AMP (100 nM) in the presence of model impurity analytes (CTP, UTP,
and GTP, all 1.0 .mu.M) and (b) MALDI spectrum of AMP in the
presence of model impurities after cleanup using the
aptamer-functionalized microchip device.
[0047] FIG. 26 depicts purifying AMP (100 nM) from a
salt-contaminated buffer solution for enhanced MALDI-MS detection
according to an embodiment of the described subject matter. (a)
depicts a spectrum of sample prior to purification using the
microchip. (b) depicts a spectrum obtained after sample
purification using an aptamer-functionalized microchip.
[0048] FIG. 27 illustrates examples incorporating the principle of
microfluidic characterization of temperature dependent biomolecular
binding: (a) A sample of specific and nonspecific reference ligands
are introduced to a receptor functionalized solid surface. (b)
After incubation at a selected temperature (controlled by
integrated heaters on the surface), a certain amount of specific
ligands bind to the receptor leaving unbound ligands and the
reference ligands in solution. (c) Similarly, a sample of specific
ligands previously bound to the receptor surface can be released
(d) following modification of the surface temperature above or
below a binding temperature. (e) The sample is transferred from the
surfaces to the MALDI plate, via an integrated microdevice, for MS
analysis.
[0049] FIG. 28 depicts a schematic of the microfluidic device of an
example embodiment used for MALDI-MS based characterization of
temperature dependent aptamer-protein binding.
[0050] FIG. 29 depicts temperature dependent binding of AMP to
anti-AMP aptamer according to an embodiment of the described
subject matter. GMP standard is of equal concentration to AMP for
each sample.
[0051] FIG. 30 depicts temperature dependent binding of PDGF to
anti-PDGF aptamer obtained similarly to the protocol used for the
AMP device according to an embodiment of the described subject
matter. VEGF utilized as a standard.
[0052] FIG. 31 depicts temperature dependent binding of
spiegelmer-vasopressin according to an embodiment of the described
subject matter. P18 standard is of equal concentration to the
spiegelmer for each sample.
[0053] FIG. 32 depicts (a) a schematic of the microfluidic
aptasensor of an embodiment of the described subject matter; (b) a
cross-sectional view along line A A' from (a) illustrating the
device's layered structure; (c) A photograph of a packaged device
of an embodiment of the described subject matter.
[0054] FIG. 33 depicts example demonstrations for illustrating the
described subject matter; 1 .mu.M sample of TMR-AVP and TO-AMP are
introduced into the aptasensor microchamber containing bare beads
and then subsequently AVP-specific aptamer.
[0055] FIG. 34 depicts time resolved fluorescence measurements for
the binding of TMR-AVP to the aptamer for an embodiment of the
described subject matter.
[0056] FIG. 35 depicts concentration dependent fluorescence
response of TMR-AVP at varying concentrations. A sample was
injected into and incubated in the extraction chamber. At this
point, TMR emission is measured from the objective lens and
recorded. A dose dependent relationship is observed.
[0057] FIG. 36 depicts enrichment by continuous infusion of a
dilute sample (100 pM) of TMR-AVP according to an embodiment of the
described subject matter. The red dashed line indicates the
relative fluorescence of a 100 nM sample. This demonstration
highlights the capability of enrichment prior to detection for
enhanced signal acquisition.
[0058] FIG. 37 depicts thermally activated release of captured
TMR-AVP according to an embodiment of the described subject matter.
An initial 1 .mu.M sample of TMR AVP is captured from solution at
.about.35.degree. C. Subsequently, the temperature is raised
incrementally while introducing pure AVP-buffer. The initiation of
a sharp decrease in signal at .about.46.degree. C. can be observed,
continuing to vanish as the temperature increases, demonstrating
release of the TMR-AVP from the aptamer.
[0059] FIG. 38 illustrates detection of unlabeled AVP using an
aptasensor of an embodiment of the described subject matter. A
sample of AVP is introduced into the aptasensor for capture inside
the microchamber, followed by thermally induced isocratic elution
into a pure matrix plug that is subsequently spotted onto the MALDI
plate. Mass spectra of: (a) 1 pM AVP; (b) 10 pM; (c) 100 pM; and 1
nM (d) are depicted.
[0060] FIG. 39 depicts enrichment of dilute AVP samples by
continuous infusion of prior to MALDI-MS detection: (a) 1; (b) 10;
and 100 pM (c). The ability to increase the relative signal
intensity of the molecular ion peak for AVP for ultralow
concentration samples demonstrates the utility of a microfluidic
enrichment process of an embodiment of the described subject
matter.
[0061] The presently described subject matter will now be described
in detail with reference to the Figures in connection with the
illustrative embodiments.
DETAILED DESCRIPTION
[0062] The described subject matter includes techniques and
components for minimally invasive, selective capture and release of
analytes. An aptamer is selected for its binding affinity with a
particular analyte(s). The aptamer is functionalized on a solid
phase, for example, microbeads, polymer monolith, microfabricated
supports, etc. The analyte is allowed to bind to the aptamer, for
example, in a microchamber. Once the analyte has been bound, a
temperature control sets the temperature to an appropriate
temperature at which the captured analyte is released.
[0063] Affinity binding includes the reaction between a ligand and
a specific receptor, such as an antigen and antibody or enzyme and
substrate. The strong specificity stems from the ligand and
receptor being ideally suited to one another both electrostatically
and spatially. Additionally, ligand and receptor binding can be
reversed by such stimuli as heat and ionic strength. While
antibody/lectin devices are one affinity pair, high-affinity
aptamers (e.g., an oligonucleotide that binds specifically to an
analyte via affinity interaction) derived from nucleic acid are
drawing increased attention because they can be synthesized
selectively towards any target molecule. They offer long-term
stability, relatively straightforward synthesis, and the capability
of modifiable end-chains to facilitate labeling or immobilization.
Also, aptamers can reversibly bind to their targets within an
aqueous environment, eliminating exposure of sensitive devices to
harsh reagents.
[0064] In some embodiments, the analyte can exist in an impure
form, i.e., mixed with one or more impurities. The techniques of
the described subject matter can be used to increase the PC of the
analyte. Once the analyte is allowed to bind to the aptamers
functionalized on the solid phases, the components are washed to
remove any excess impurities. Analytes from another impure complex
are allowed to bind to the aptamers, and another washing takes
place. This procedure is repeated until the desired concentration
is reached. The temperature is then set such that the concentrated
analyte is released.
[0065] In some embodiments, the described subject matter includes
collection and detection components, for example, a surface
tension-based microvalve for releasing the analyte onto a detection
surface and a mass spectrometer used to measure the analyte. Other
detection techniques include detectors for measuring the amount of
fluorescence given off by a sample of an analyte coupled with
fluorescing materials, electrospray ionization mass spectroscopy,
nuclear magnetic resonance, electrochemical techniques, impedance
techniques, and the like.
[0066] Furthermore, the use of selective release of the analyte
from the aptamers in a minimally invasive manner allows the
aptamers to be reused. Minimally invasive release also causes less
harm to the analyte.
[0067] In some embodiments, the particular aptamer/analyte binding
can cause an otherwise actively interactive analyte to be
temporarily inactivated. The analyte can be delivered to a target
location where its interactivity is desired. Selective release can
then release the analyte, which can regain the analyte's original
interactivity. For example, certain drugs are inactivated through
aptamer binding and can be targeted to specific body locations for
the drug to take effect.
[0068] Analytes include any appropriate biochemical component,
biomolecule, pharmaceutical, protein, nucleotide sequence, cell,
virus, compound, or the like. For example, analytes include toxic
molecules, compounds, or bacteria, viruses, or the like, which can
appear in pharmaceuticals, food, or the like. Principles of the
described subject matter can be used to selectively capture these
toxins and release the toxins in a safe environment. In other
embodiments, aptamers can bind to peptides, proteins, small
molecules, other inorganic and organic molecules, cells, viruses,
micro organisms, and the like. It should be noted that analytes can
be used beyond components merely for analytical purposes. Any
suitable component which is selectively captured and released by an
aptamer is encompassed within the described subject matter. For
example, selective capture and targeted release of analytes can be
used for drug delivery. Also, captured analytes can be permanently
bound to an aptamer, such as in a technique for removing
biochemical hazards from the environment. Furthermore, analytes can
be inactivated when attached to an aptamer, such as in techniques
for reducing the effect of harmful chemicals. Still further,
analytes can have their properties changed as a result of being
bound to an aptamer, thereby producing a secondary effect of the
analyte as desired.
[0069] In one embodiment, the described subject matter includes a
microfluidic device that accomplishes integrated, all-aqueous
realization of specific extraction, concentration, and coupling to
mass spectrometric detection of biomolecular analytes. The device
uses an aptamer functionalized on microbeads to achieve highly
selective analyte capture and concentration. By on-chip temperature
control, the device makes novel use of thermally induced,
reversible breakage of the analyte-aptamer complex at low
temperature (38.degree. C.) to release the captured analyte and
regenerate microbead surfaces. Furthermore, using a hydrophobic
microvalve, the released analyte is directly spotted onto an
analysis plate for detection by MALDI-TOF mass spectrometry.
[0070] In another embodiment, a microfluidic device is used for PC
and release of specific analytes. The device surfaces are
functionalized with an RNA aptamer that selectively binds a target
analyte. The device employs thermally induced denaturing of the
aptamer for intelligent release. This occurs at .about.32.5.degree.
C., a safe temperature for thermally sensitive analytes and ligands
functionalizing the device surface. Since denaturing the aptamer is
reversible, this permits reuse. In addition, operation is
simplified as analyte capture and release occur in aqueous medium
without altering solvent composition or polarity. Although
applicable to many analytes, we use a model analyte, adenosine
monophosphate (AMP).
[0071] In one embodiment, the device includes a microchamber packed
with aptamer-immobilized microbeads for analyte PC, a microheater
and temperature sensor for thermally induced analyte release, and
microchannels equipped with a passive valve using surface tension
for spotting the released analyte onto a MALDI analysis plate (FIG.
14). Analyte, matrix, and wash solutions are introduced via a
sample inlet 1400. The bead inlet facilitates packing the aptamer
chamber 1404 with microbeads. A resistive heater and temperature
sensor 1410 are placed below aptamer chamber 1404 to promote
efficient heating and accurate sensing. The valve and deposition
well 1406 are placed near the aptamer chamber 1404 to reduce
analyte dilution after release due to adsorption to the channel
walls or diffusion to dead fluid volumes. A waste outlet 1408 is
used to remove any excess fluids or impurities. A heater 1410 is
used to set the temperature of the chamber to an appropriate
thermal release temperature.
[0072] The microfluidic chip structure is realized with three
sandwiched polymer layers. Layer 1412 incorporates the inlets,
passive valve, and waste outlet. To reduce bubble entrapment or
dead volumes during sample spotting, layer 1414 provides an air
vent connected to the spotting well. It also encapsulates the
fluidic network present in layer 1412. Layer 1416 defines the
spotting well and houses an air vent channel. A vent 1418 is used
to prevent dead air volume during spotting. The sample is deposited
on to a MALDI plate 1420 for analysis.
[0073] To illustrate some principles of the described subject
matter, PC is achieved with adenosine monophosphate (AMP) as a
model analyte by use of an adenosine triphosphate aptamer
(ATP-aptamer) on an integrated microfluidic device. The device is
coupled to a matrix assisted laser desorption/ionization mass
spectrometry (MALDI-MS) machine where AMP is analyzed.
[0074] AMP is introduced into the microchamber and extracted by the
aptamer. A rinse follows to flush out impurities through the waste
outlet. For concentration, this procedure can be repeated to
saturate AMP on the beads. Next, the microchamber is heated using
the microheater to reverse the AMP/ATP-aptamer bond. This releases
the analyte from the beads. In order to direct flow of a released
AMP sample through a secondary channel leading to the spotting
well, a valve based on surface tension is used.
[0075] The passive microfluidic valve, which directs the released
analyte to the spotting outlet, exploits surface tension. That is,
a pressure difference exists at the air-liquid interface in a
sudden narrowing of a microchannel with hydrophobic surfaces. This
pressure difference is provided by the Young-Laplace relationship
and serves as a pressure barrier (e.g., critical pressure), which,
only when exceeded, will allow the eluent (e.g., eluted sample) to
enter the secondary channel and the spotting outlet:
.DELTA. p = 2 .gamma. cos ( .theta. c ) [ ( 1 w 1 + 1 h 1 ) - ( 1 w
2 + 1 h 2 ) ] ( 1 ) ##EQU00001##
[0076] In Eq. 1, .gamma., .theta..sub.c, w, and h are the surface
energy, contact angle, width, and height of the channel,
respectively, at the air-liquid interface. This pressure drop
allows the hydrophobic channel to act as a passive valve, and, in
the exemplary device, is used to regulate flow between the spotting
outlet and the waste outlet. Since the packed chamber is the
primary flow resister in the device (FIG. 14), a modified
Poiseuille equation is used to determine its pressure drop:
.DELTA. p = 150 .eta. u ( 1 - ) 2 L d 0 2 2 ( 2 ) ##EQU00002##
[0077] Here, n, u, L, .epsilon., and d.sub.o represent the dynamic
viscosity, average fluid velocity, channel length, void fraction,
and bead diameter, respectively. After sample spotting, the chip is
removed from the MALDI plate for analysis.
Example 1
[0078] One embodiment of the described subject matter further
demonstrates some of the principles described. Biotinylated
ATP-aptamer is purified while AMP, cytidine, uridine, and guanosine
triphosphate (C/U/G-TP) are synthesized. The matrix solution is
prepared from 2,4,6-trihydroxy-acetophenone (2,4,6-THAP),
2,3,4-THAP, and diammonium citrate at 0.1, 0.05, and 0.075 M
concentrations, respectively, in a 3:5 (v/v) mixture of
acetonitrile/water. Streptavidin coated agarose beads (.about.50
.mu.m OD) provide support surfaces while a Voyager-DE time of
flight mass spectrometer (Applied Biosystems) is used for mass
analysis. DNA grade water is used in the example.
[0079] The device fabrication process is shown in FIG. 15. FIG. 15
depicts an example fabrication process flow as seen from
cross-section A-A' in FIG. 14. FIG. 15a depicts PR patterning for
Cr/Au deposition. FIG. 15b depicts thermal evaporation of a Cr/Au
bi-layer. FIG. 15c depicts lift-off patterning of Cr/Au and PECVD
deposition of SiO.sub.2. FIG. 15d depicts a substrate drilled for
fluidic ports and 3 through-hole polydimethylsiloxane (PDMS) layers
aligned and permanently bonded. FIG. 15e depicts a packaged chip
with tubing.
[0080] SU-8 molds for each microfluidic layer are first created,
with which PDMS prepolymer is cast into an in-house built
through-hole PDMS sandwiching jig and cured (60.degree. C. for 8
hours). Meanwhile, Cr/Au (5/100 nm) films are deposited, patterned,
and passivated with SiO.sub.2 on glass substrates, realizing the
microheater and temperature sensor. Following plasma (O.sub.2)
treatment of each bonding interface, all three PDMS layers and the
glass substrate are then aligned using optical microscopy and an
x-y-z stage before permanently bonding them to each other
consecutively. Finally, microbeads are packed into the aptamer
chamber and the entire assembly is subsequently attached to a MALDI
plate via spontaneous adhesion.
[0081] The device is first rinsed with water (10 .mu.l/min, 10 min)
All following washing and loading schemes are identical.
ATP-aptamer is loaded (10 .mu.M, 10 .mu.l, 20 min) into the chamber
to functionalize the bead bed. After a subsequent wash, a pure
matrix mass spectrum (MS) is acquired for a negative control.
[0082] An arbitrary concentration of fluorescein solution is used
to characterize the valving operation and sample spot
characteristics. For valving demonstrations, solution is first
flowed through zone 1 (10 .mu.l/min) below the critical pressure of
the valve. To operate the passive valve, the waste outlet is
plugged while maintaining a constant flow rate. This increases the
pressure in the flow stream adjacent to the valve to eventually
overcome its critical pressure and accentuate it. A 20.times.
microscope objective is focused on the valve area during
demonstration. To test sample spot characteristics, the waste
stream is plugged while fluorescent solution is deposited from the
chip using different flow rates (10-50 .mu.l/min). Each spot is
recorded and analyzed using a 20.times. objective.
[0083] For extraction/purification, 0.1 and 1.0 .mu.M AMP samples
are loaded into the aptamer chamber separately. A rinse follows to
eliminate non-specific compounds. AMP is then released from the
aptamer by raising the chamber temperature to 38.degree. C. while
introducing a matrix sample plug. The sample/matrix plug is then
transferred to the spotting well and deposited onto the MALDI plate
to be subsequently analyzed. Similarly, for specific extraction of
AMP, a solution of AMP, CTP, UTP, and GTP (1 .mu.M) is loaded into
the aptamer chamber. After an incubation (5 min) and wash procedure
(to flush out non-target molecules), matrix is loaded into the
chamber. The heater is activated to release the molecules currently
on the aptamer and deposit them onto the MALDI plate for
analysis.
[0084] For preconcentration of AMP, a multiple injection scheme is
used. The aptamer chamber is consecutively loaded with 10 nM
injections of AMP sample. Each injection is incubated (5 min) and
followed by a rinse. Upon suspected saturation of the aptamer with
AMP, the chamber is heated to release the analyte into a matrix
plug. The analyte is then deposited for analysis.
[0085] To ensure the validity of the higher-level data, properties
of the microfluidic valve (FIG. 16) are obtained. At a steady flow
rate, the pressure difference imparted by the microfluidic valve
1602 impinges fluid access to the spotting outlet. When the waste
outlet 1600 is open and at flow rates below 50 .mu.l/min (e.g., 10
.mu.l/min), fluid flow bypasses the valve since the hydrodynamic
pressure driving flow (.about.686 Pa) was smaller than the critical
pressure of the valve (FIG. 16a). To direct flow to the MALDI
plate, the pressure drop between the sample inlets to the waste
outlet 1600 can be greater than the valve's critical pressure
(i.e., above 3.154 kPa). This is accomplished by plugging the waste
outlet 1600 using an external valve, and maintaining a constant
flow rate, which allows fluorescein solution to enter the channel
leading to the spotting outlet (FIG. 16b).
[0086] Sample spot size can be a useful characteristic during MALDI
analysis. Large volume spots can promote dissociation of matrix
from sample upon spot crystallization, resulting in poor
ionization. Additionally, non-uniformity in sample concentration
throughout the spot can occur, degrading analysis. Spot size
produced by the device is measured as a function of driving flow
rate (FIG. 17). For low flow rates (10-30 .mu.l/min), spot sizes
approximately equal to the well size are obtained (.about.500
.mu.m). Higher flow rates (>40 .mu.l/min) generate a larger spot
diameter (.about.700-800 .mu.m) since the seal between the PDMS and
MALDI plate at the location of the spotting well tends to falter at
the resulting higher pressures. Consequently, the sample spot
broadens once the chip is removed from the plate to obtain a spot
size. However, this is of no detriment to the overall performance
of the device compared to conventional spotting (with syringe or
pipette), where crystallized spots are larger (>1 mm).
[0087] As described, to demonstrate AMP extraction by ATP-aptamer,
two sample solutions of AMP (0.1 & 1.0 .mu.M) are first
injected into the chamber. AMP is released and deposited onto a
MALDI-MS plate and analyzed (FIG. 18). The MS of a spot obtained
from a 0.1 .mu.M AMP solution (FIG. 18a) shows a distinctive mass
peak of 348.11 Da, which corresponds to AMP (established value:
347.22 Da). Since AMP concentration is relatively low, the
magnitude of this peak is comparable to several peaks from the
MALDI matrix (338, 393, & 468 Da). A mass spectrum obtained
from a 1.0 .mu.M AMP solution (FIG. 18b) improves the
analyte-to-reference peak contrast. In this case, the AMP peak
dominates reference peak amplitudes, suggesting that concentrating
dilute samples can improve analyte recognition. Although mass
spectrometry is a precise detection technique, various fluctuations
in instrument settings (e.g., electromagnetic field strength,
detector vibrations, and laser intensity) will cause expected m/z
values of a substance to vary slightly. Hence, the molecular ion
peak in m/z for AMP (and other noteworthy peaks) in this
description will not always be exactly their predicted value (e.g.,
348.22 for AMP) and would rather deviate slightly. Such slight
deviations do not affect molecular identification and are generally
accepted for mass spectrometry.
[0088] In some embodiments, purification of analytes can be a
valuable tool for selectively controlling analytes in biochemical
applications. AMP is selectively extracted from a homogeneous
solution of AMP, CTP, UTP, and GTP (1.0 .mu.M each) by loading the
sample into the aptamer chamber and subsequently washing the
chamber to isolate AMP. A deposited sample spot is obtained
similarly to previous protocol. FIG. 19 represents the MS of an
analyte sample originating from the homogeneous solution. The ratio
of AMP to noise is comparable to that seen in FIG. 18b, where only
AMP is present in the solution. Additional non-target peaks are
observed (480, 484, & 523 Da). However, their intensities are
significantly lower than the AMP peak, suggesting that the amount
of non-specific binding is negligible. This confirms the ability of
the described subject matter to selectively extract and concentrate
biomolecules for analytical applications.
[0089] As a sample preparatory technique, PC can be useful for
sample conditioning and analyte signal improvement. In another
embodiment, PC performance of the device is demonstrated by loading
a dilute AMP sample into the aptamer chamber multiple times to
saturate the analyte on the aptamer bed before release for MS
analysis. Dilute sample concentration is chosen to be lower
(.about.0.01 .mu.M) in order to highlight the detection enhancement
due to PC. 25 consecutive dilute AMP samples are injected into the
aptamer chamber, release the captured AMP with heat, and transfer
the concentrated plug to the spotting well. An MS is obtained from
the resulting sample spot (FIG. 20a). An AMP peak to noise ratio
slightly higher than that seen in FIG. 18a is observed,
demonstrating the successful concentration of AMP by
.about.10.times..
[0090] More consecutive injections of dilute AMP solution are
attempted to obtain the maximum PC factor of the device. A maximum
of 250 injections are performed. Following the final injection, a
sample spot is obtained and analyzed with MALDI-MS, similar to the
protocol with 25 injections (FIG. 20b). It can be seen that he AMP
peak dominates those of reference peaks and the AMP peak to noise
peak ratio is comparable to that shown in FIG. 18b. This suggests a
PC factor of nearly 100.times.. This is a useful PC factor, similar
to that seen in the reverse-phase devices, but with the advantage
of higher specificity. AMP sample injections are stopped after 250
injections due to demonstration practicality, not because of actual
saturation of the analyte. This suggests the possibility for larger
PC factors using principles of the described subject matter.
Example 2
[0091] Another embodiment illustrates the principles of the
described subject matter. Biotinylated adenosine triphosphate
aptamer (bio-ATP-40-1, or ATP-aptamer) is HPLC purified by
Integrated DNA Tech. AMP is synthesized and fluorescently labeled
with thiazole orange (TO). Buffer solution (pH 7.4) is prepared
from Tris-HCl (20 mM), NaCl (140 mM), KCl (5 mM), and MgCl.sub.2 (5
mM) in water. Streptavidin coated polystyrene beads (50-80 .mu.m,
OD) are acquired from Pierce. A Nikon Eclipse TE300 microscope and
CCD is employed for fluorescence detection. Temperature control is
accomplished with a thermoelectric device and type-K thermocouple.
A New Era NE-1000 syringe pump enables flow in the device.
[0092] A device schematic is shown in FIG. 1. Channels 100 and 102
(5.1 mm.times.400 .mu.m.times.40 .mu.m) transferred sample and
discharged waste from the chamber 112 (8.7 mm.times.3 mm.times.140
.mu.m). Microbead 114 packing into the chamber is accomplished
through 104. Ports 106, 108, and 110 are 1 mm in radius and 140
.mu.m thick. Chamber and microfluidic network volumes,
respectively, are 3.09 .mu.l and 3.60 .mu.l.
[0093] Channels are fabricated using PDMS micro-molding by soft
lithography (FIG. 2). A mold is created on a 4-in silicon wafer by
patterning SU-8. PDMS pre-polymer solution is mixed (10:1; w:w),
degassed, and semi-cured (70.degree. C., 50 min) over the mold
(FIG. 2a-b). In parallel, glass substrates are cleaved (25
mm.times.30 mm) and drilled to create ports 106-110 (refer to FIG.
1) (FIG. 2c-e). The semi-cured PDMS sheet is removed from the mold,
aligned, and bonded to the glass following O.sub.2 plasma treatment
of the bonding interface. Permanent bonding is realized with a
final bake (25 min at 85.degree. C.).
[0094] Packaging of the device is accomplished by inserting silica
capillary and Tygon tubing (FIG. 2f), (0.6 mm ID, 0.7 mm OD) and
(0.6 mm ID, 3.18 OD), respectively into ports 106-110. The
interfaces are then sealed with epoxy.
[0095] The device is mounted on the microscope stage using clips or
double-sided tape. A blue excitation filter combined with a
green-pass dichroic mirror is used. A 10.times. objective is kept
focused on a single area of the chamber.
[0096] The chamber is initially rinsed with buffer (50 .mu.l/min,
10 min). The following rinses are identical. Streptavidin coated
beads are introduced via c3 by manual pressure. The chamber and
channels are rinsed and bio-ATP-aptamer is injected (20 .mu.M, 20
.mu.l, 10 .mu.l/min) and incubated (20 min) in the chamber. After a
final rinse, a fluorescence control is established.
[0097] Extracting distinct concentrations of AMP (24.5.degree. C.,
10 .mu.l, 10 .mu.l/min) establishes a fluorescence intensity curve.
The procedures use the above injection parameters. Solution
concentrations range from 0.1-10 .mu.M and fluorescence is detected
after rinsing between separate extractions.
[0098] For PC of AMP, multiple solution injections are used. Two
devices (Device 1 & Device 2) are consecutively loaded with 200
nM and 500 nM injections, respectively. On either device, each
injection is incubated (5 min), rinsed, and checked for
fluorescence before the next injection occurs.
[0099] To estimate the relationship between fluorescence signal
intensity and surface concentration, AMP solution is extracted at
increasing concentrations onto multiple devices. An S-shaped
relationship can be observed between the mean fluorescence
intensity and AMP concentration, which appears to be a
dose-responsive characteristic (FIG. 3).
[0100] Preconcentration is demonstrated with 2 dilute solutions of
TO-AMP on separate devices by extracting multiple injections on
each device (FIG. 4). In both demonstrations, fluorescence signal
increases after each consecutive sample load, indicating increased
concentration of bound TO-AMP on the surface. In addition, Devices
1 and 2, after a roughly 10-fold PC, show no sign of signal
saturation within the tested injection range, meaning the surface
is capable of concentrating yet more analyte.
[0101] To demonstrate the aptamer thermal release properties, a 10
.mu.M AMP solution is extracted and eluted for a range of
temperatures (30-50.degree. C.) (FIG. 5). After extraction of AMP
on the aptamer surface, a high intensity fluorescence signal is
obtained. At 32.5.degree. C., there is a sharp decrease in signal
intensity (near baseline). As the temperature is further increased
to 47.5.degree. C., the signal matches the baseline intensity. No
signal implies an absence of coupled AMP on the device affinity
surface, meaning release of analyte. Thus, the device exhibits
adequate release of a captured target analyte at sufficiently low
temperature (32.5.degree. C.). Regeneration at this temperature
does not endanger the viability of thermally sensitive
biomolecules.
[0102] To demonstrate the functionality of the aptamer surface
post-release of AMP, extraction of 10 .mu.M AMP (1.sup.st
Extraction) is followed by three elevated temperature release 75,
85, and 95.degree. C., which is in turn followed by a second
extraction (2.sup.nd Extraction) (FIG. 6 series A, B, and C,
respectively). It can be observed that when TO-AMP was released at
75.degree. C. in series A, the subsequent extraction (the 2.sup.nd
Extraction) yielded fluorescence intensity comparable to that from
the pre-release extraction. On the other hand, in series B and C,
2.sup.nd Extraction fluorescence signals were significantly lower
following release at further elevated temperatures (85 and
95.degree. C., respectively). This led us to conclude that most
aptamer molecules had separated from the microbeads because of
streptavidin-biotin denaturation.
Example 3
[0103] Biotinylated adenosine triphosphate aptamer (bio-ATP 40-1,
or ATP-aptamer) (FIG. 7a) is acquired (e.g., from Integrated DNA
Technologies (Coralville, Iowa)) and purified by high pressure
liquid chromatography. AMP is coupled with thiazole orange
("TO-AMP") (FIG. 7b). To-AMP can be replaced with any appropriate
molecule+probe combination. Adenosine triphosphate (ATP) is
purchased from Sigma-Aldrich Co. (Milwaukee, Wis.). Diethyl
pyrocarbonate treated sterile water (SW), from Fisher (Pittsburgh,
Pa.), is used. Buffer solution (pH 7.4) is prepared by mixing
Tris-HCl (20 mM), NaCl (140 mM), KCl (5 mM), and MgCl.sub.2 (5 mM)
in sterile water. Chemicals for the buffer solution are purchased
through Fisher Scientific. ATP aptamer, TO-AMP, and ATP working
solutions are all prepared using Tris-HCl buffer. UltraLink
immobilized streptavidin polystyrene beads (50-80 .mu.m in
diameter) are acquired from Pierce (Rockford, Ill.). All solvents,
isopropyl alcohol (IPA), methyl alcohol, and acetone are of
purified grade (Mallinekrodt Baker, Phillipsburg, N.J.). SU-8 2025
and 2100 is purchased from MicroChem (Newton, Mass.).
Poly-dimethylsiloxane (PDMS) is acquired from Robert McKeown
Company (Somerville, N.J.). Torr Seal epoxy and silicone glue is
obtained from Varian (Palo Alto, Calif.) and Action Electronics
(Santa Ana, Calif.), respectively. Glass slides (25 mm.times.75 mm)
are purchased from Fisher. Silica capillary tubing and Tygon
poly-vinyl chloride (PVC) tubing are purchased from Polymicro
Technologies (Phoenix, Ariz.) and McMaster Carr (Dayton, N.J.),
respectively. Arctic Silver 5 is obtained from Arctic Silver Inc.
(e.g., used for IC component bonding) and Kapton Tape is purchased
from Techni-Tool (Worcester, Pa.).
[0104] Mercury vapor lamp induced fluorescence using a Nikon
Eclipse TE300 inverted epi-fluorescence microscope (Nikon, USA) is
employed for detection. Fluorescence micrographs are recorded using
a Q-Imaging model Retiga 2000R Mono-12-bit CCD and analyzed with
Q-Capture Pro software (Austin, Tex.). Device temperature control
is performed using a thermoelectric device from Meteor (model:
CP1.4-71-06L, Trenton, N.J.). DC potential is supplied to the
thermoelectric device with an Agilent E3631 DC power supply (Santa
Clara, Calif.). A type-K surface thermocouple model CO3-K and a
model HHM-290 multimeter (Omega, Stamford, Conn.) are used to
measure device temperature. Microfluidic flow is provided from a
New Era model NE-1000 syringe pump (Farmingdale, N.Y.), 5 cm.sup.3
syringes, and 21 gauge (38.1 mm long) needles (Becton Dickinson,
Franklin Lakes, N.J.). Diamond-tipped drill bits (0.7 mm diameter)
and a Model 7000 standard drill press are obtained from Servo
Products (Eastlake, Ohio).
[0105] A device is shown in FIG. 8. The channels are numbered for
reference. Channels 800 and 802 (5.1 mm.times.400 .mu.m.times.40
.mu.m) are used to deliver sample and buffer solution to the
chamber (8.7 mm.times.3 mm.times.140 .mu.m). Channel 804 is used to
pack the beads 814 (e.g., polystyrene beads). The ports have radii
of 1 mm each and are 140 .mu.m thick. Hence, chamber 812 has an
effective volume of 3.09 micro-liters with the tapers taken into
consideration, whereas the microfluidic device volume (on-chip) is
3.60 micro-liters. Using Poiseuille-flow, the maximum pressure drop
across this device (port to port), excluding beads, can be
calculated from:
Q = .pi. ( D h ) 4 .DELTA. p 128 .mu. l . ( 3 ) ##EQU00003##
[0106] Here, Q is the flow rate, .DELTA..rho. is the pressure drop,
.mu. is the dynamic viscosity of the fluid, l is the channel
length, and D.sub.h is the hydraulic diameter given by the
expression
D h = 4 A P . ( 4 ) ##EQU00004##
[0107] In (4), A is the cross-sectional area of the channel and P
is the wetted perimeter. For water, the calculated pressure drop
for Q=50 .mu.l/min used in the demonstrations is 6.83 kilo-Pascal.
When considering a packed chamber of micro-beads, the pressure
increase is estimated to be 10-20 times greater.
[0108] Microchip solid-phase extraction (SPE) devices are
fabricated on glass slides by PDMS micro-molding using standard
soft lithography techniques. A simplified device process flow (FIG.
9) shows primary fabrication procedures. An SU-8 mold for PDMS
curing and channel fabrication is created on silicon wafers (101
mm) from Silicon Quest International (Santa Clara, Calif.).
Fabrication begins with deposition and patterning of 15 nm Cr
alignment marks via thermal evaporation on an Edwards/BOC Auto306
thermal evaporator (Wilmington, Mass.), followed by lift-off in
acetone overnight. Secondly, patterning of SU-8 2025 realizes
channels 800 and 802 (40 .mu.m thick) (FIG. 9b), whereas SU-8 2100
resist completes the mold, producing the reaction chamber and
channel 804 (140 .mu.m).
[0109] PDMS pre-polymer solution is mixed with a mass ratio of 10:1
and distributed on the mold (FIG. 9c). The pre-polymer is degassed
by vacuum (30 min) and followed by semi-curing (70.degree. C., 50
min) In parallel, glass substrates are diced (25 mm.times.30 mm)
and drilled to create the access ports (806-810) (FIG. 9d). The
glass substrates are then cleaned using a solution of
H.sub.2SO.sub.4 and H.sub.2O.sub.2 (7:4 vol/vol at 130.degree. C.).
In other embodiments, ports can be fabricated in the PDMS blank
layer. The semi-cured PDMS sheet is removed from the SU-8 mold,
aligned and bonded to the glass slides following O.sub.2 plasma
treatment of the bonding interface in a Technics Series 800 Micro
R1E device (100 mtorr and 85 W) for 15 seconds. Permanent bonding
and curing of PDMS to the substrate is performed by heating the
chip (25 min at 85.degree. C.).
[0110] Packaging is accomplished by inserting a combination of
silica capillary tubing (0.6 mm ID, 0.7 mm OD) segments along with
Tygon PVC tubing (0.6 mm ID, 3.18 OD) through the drilled access
ports (FIG. 9e). The connection interfaces are sealed using
silicone glue and Torr seal epoxy. For thermal related
demonstrations, a thermocouple is subsequently sandwiched between a
peltier device and the bottom of the microfluidic chip (FIG. 90.
The components are held together by thermal interfacing paste or
Kapton Tape.
[0111] Fluorescence detection is done using a Nikon TE300 and a
Q-Imaging Retiga 2000R device. During analyte binding, TO emission
occurs at 530 nm when excited at 480 nm, so a blue light filter and
green-pass dichroic mirror are used accordingly. The device is
mounted in the same position using double-sided scotch tape marks
on the microscope stage. For each image, a 10.times. objective is
used to collect emitted fluorophores from the same area of the
chamber (FIG. 10). These operating conditions are identical for all
images taken for fluorescence detection of the analyte.
[0112] The entire microfluidic device is flushed (50 .mu.l/min)
with the buffer solution for 30 minutes by using any port as an
inlet and collecting waste from both remaining ports. Streptavidin
coated beads are suspended in buffer (4 ml) and loaded into a 5 ml
syringe. Manual pressure is used to pack the beads from channel 804
via port 810 into the chamber. Subsequently, channel 804 is sealed
permanently near the port interface using silicone glue. The
chamber and channels are washed (50 .mu.l/min) with buffer (30 min)
through 800. ATP-aptamer solution (20 .mu.l, 20 .mu.M) is injected
(10 .mu.l/min) and allowed to incubate (20 min) in the chamber. The
channel is washed again (50 .mu.l/min for 20 min) and a baseline
fluorescence signal is taken.
[0113] For SP purification/extraction, TO-AMP at different
concentrations (400 nM, 500 nM and 10 .mu.M) is loaded (10 .mu.l at
10 .mu.l/min) into the reaction chamber from channel 800. The
solution is kept stagnant in the chamber for 10-15 minutes to allow
complete interaction between the analyte and aptamer surface of the
beads. Following the purification of analytes, the chamber is
washed (50 .mu.l/min for 15 min) with buffer to eliminate all
non-specific compounds, un-reacted molecules, and impurities. A
subsequent fluorescence image is taken. TO-AMP is released and
collected in two ways: the first technique uses competitive
displacement of TO-AMP by incubating different concentrations of
ATP (800 .mu.M and 3200 .mu.M); the second technique uses elevated
chip temperature (80.degree. C.) while buffer is flowed (10 .mu.l
at 5 .mu.l/min) through to collect analyte.
[0114] During purification, time resolved analyte adsorption
demonstrations are conducted. For a 400 nano-molar concentration of
TO-AMP solution, fluorescence micrographs are recorded at time
intervals of 1 minute. Images ceased to be taken after the observed
fluorescence level shows no appreciable change.
[0115] An integrated SPE bed is prepared using a double weir design
forming a cavity. Using PDMS for the channel material can present
challenges. Since PDMS is pliable, beads can be pushed under the
weir structures under positive pressure resulting in backpressure.
During bead introduction, port 810 is prone to clogging. Designs
using drilled access ports in the PDMS blank layer can be a source
of this problem. The holes in PDMS are plagued with burrs
containing loose PDMS particles not cleared while drilling, which
became obstacles and instigated clogging. This is mitigated by
generating reversed flow and allowing beads to dislodge and flow
back toward the source. On occasion, several forward/reverse
pumping cycles are used to fully clear obstructions and continue
filling the chamber. Drilling holes in the glass slides provides
smoother, burr-free edges. Using this technique, fully packed
chambers are realized in over 90 percent of devices.
[0116] Another source of backpressure comes from the beads
themselves, especially when using narrow uniform-width channels. To
minimize backpressure of this sort, a widened chamber design is
employed to contain the affinity matrix. Although the expansion
ratio utilized (400 .mu.m/3 mm) can be improved, the backpressure
is minimized, and the device functions in a fashion similar to open
micro-channels.
[0117] To determine the approximate binding time of TO-AMP
molecules to fully saturate the affinity matrix, fluorescence
micrographs in discrete time intervals (1 min) immediately
following a sample injection of TO-AMP (400 nM) are recorded.
Fluorescence intensity measurements are obtained in a straight line
direction (A-A') across each micrograph, averaged and then plotted
as a function of time (FIG. 11). Similar analysis techniques are
used to quantitate all ensuing data. No appreciable increase in
fluorescence intensity occurs after 10 minutes of incubation
time.
[0118] To determine the ability of this SPE matrix to retrieve
specific analytes, three sample solutions of TO-AMP (400 nM, 500 nM
and 10 .mu.M) are injected as described above. The solutions are
allowed to interact with the matrix (10-15 min) and the chamber is
then washed (50 .mu.l/min for 15 min) with buffer before detection
is performed. Imaging and fluorescence analysis conditions are
similar to those used in the time resolved demonstration, only
without resolving the temporal dimension. Data is presented in FIG.
12. It is useful to note that the signals are distinct and
non-overlapping for each concentration. The "noisy" nature of the
signal comes from the dark areas in between individual beads, as
shown by the micrograph insets (FIG. 12a-d), rather than actual
noise in the signal. The baseline fluorescence supports this
explanation. No other radiation wavelength is detected from the
micrographs other than that which is specific to TO emission (530
nm), further emphasizing the selective nature of the
TO-AMP/ATP-aptamer interaction.
[0119] Three different concentrations of TO-AMP solution are
injected into this device producing 3 separate normalized
fluorescence profiles, implying that gradual concentration can be
achieved over time. This data suggests a potential concentration
factor of 20 if identical injection and collection volumes are
used. Although a 10 micro-molar solution of TO-AMP is the highest
concentration used in the demonstrations, it presents no upper
limit. It is feasible that the saturation threshold of TO-AMP to
ATP-aptamer has not been breached as of yet for the packed
microfluidic matrix presented in this demonstration.
[0120] The device is capable of capturing and releasing TO-AMP
using two release techniques. The first is competitive displacement
using a concentration gradient of ATP analyte (FIG. 13a). The
second is thermal energy (FIG. 13b). After each competitive ATP
solution (800 .mu.M and 3.2 mM) injection (10 .mu.l), fluorescence
intensity is measured. Five extraction and release cycles are
performed (2 shown here) using thermal energy. Solution conditions
and sampling using TO-AMP (400 nM) closely tracks those employed in
SPE demonstrations. Fluorescence measurements are taken after each
capture and release wash procedure.
[0121] Competitive displacement is used for only one cycle since it
proves less efficient at releasing TO-AMP than thermal energy.
While thermal cycling, the extraction signal in cycle 2 deviates by
16.7 percent from that recorded during cycle 1. It is also
determined that the transition temperature used to release TO-AMP
from ATP-aptamer falls between a range (about 80-85.degree. C.). It
should be noted that the release temperature can vary according to
the analyte, aptamer, and analyte/aptamer combination used and
according to the particular purpose of the application. Initial
attempts used 60 and 95 degrees centigrade while flowing buffer
solution (2 min at 5 .mu.l/min) to collect the released analyte;
the latter causes denaturation of streptavidin and damage of the
streptavidin-biotin (SB) bond, whereas the former does not unfold
the aptamer/target bond. Even so, the use of any particular release
technique depends on the particular parameters of the application.
In some embodiments, competitive displacement can be used instead
of thermal release, and competitive displacement remains within the
scope of the described subject matter.
[0122] It should be noted that specific release temperatures can
depend on the particular analyte, aptamer, or combination of
analyte and aptamer used. In some embodiments, minimally invasive
capture and release of analytes is performed at a temperature which
is not harmful to the analyte and is at a temperature where thermal
release occurs.
[0123] In other embodiments, the temperature control can be used to
either raise or lower the temperature. For example, a resistor in
contact with the micro chamber where the analyte/aptamer complex is
located can be used to raise the temperature. In another
embodiment, a cooling mechanism, such as an air cooler, refrigerant
mechanism, or the like can be used to lower the temperature of the
analyte/aptamer complex. The specific set point at which the
aptamer/analyte bond is released can either be above the
temperature of the device (e.g., the temperature raised using a
heater) or can be lower than the temperature of the device (e.g.,
the temperature lowered using a cooling mechanism). The specific
parameters and needs of the application can dictate the specific
temperature shift needed for thermal release of the analyte.
[0124] Another embodiment demonstrates the principles of the
described techniques. A microfluidic apparatus achieves specific
extraction, concentration, and isocratic elution of biomolecular
analytes with coupling to label-free mass spectrometric detection.
Analytes in a liquid phase are specifically captured and
concentrated via their affinity binding to aptamers, which are
immobilized on microbeads packed inside a microchamber. Exploiting
thermally induced, reversible disruption of aptamer-analyte binding
via on-chip temperature control with an integrated heater and
temperature sensor, the captured analytes are released into the
liquid phase, and then isocratically eluted and transferred via a
microvalve for detection via matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS). The
functionality of the apparatus is demonstrated using adenosine
monophosphate (AMP) as a model analyte. Results indicate that the
device is capable of purifying and concentrating the analyte from a
sample mixed with non-specific analytes and contaminated with
salts. In addition, thermally induced analyte release is performed
at one or more temperatures (e.g., 45.degree. C.), and mass spectra
obtained from MALDI-MS demonstrate detection of AMP at
concentrations as low as 10 nM.
[0125] Some embodiments illustrate a microfluidic approach intended
to improve SPE sample preparation for MALDI-MS. Using a
miniaturized microchip, which is coupled to a MALDI-MS analysis
plate, selective purification, enrichment, and enhanced label-free
detection of trace amounts of a biomolecular analyte via
aptamer-functionalized surfaces is described. This is possible due
to affinity interaction between an analyte and an aptamer molecule,
allowing highly discriminate purification. Moreover, exploiting the
strong temperature dependence of the aptamer-analyte binding,
recovery of the purified analyte and regeneration of the aptamer
receptor are possible by a modest temperature increase (45.degree.
C.). This allows the analyte molecules to be eluted in a single
aqueous phase; that is, isocratic elution is accomplished. The
described device includes a microchamber packed with
aptamer-functionalized microbeads for analyte extraction and
purification, a microheater and temperature sensor for thermally
induced analyte release, and microchannels in conjunction with a
surface tension-based valve for the control of sample flow. The
microfluidic chip is interfaced to a standard MALDI-MS analysis
plate for mass quantification. Using the metabolic hormone
adenosine monophosphate (AMP) as a model analyte, detection of AMP
at varying concentrations, consecutive infusion of trace AMP to
concentrate and purify the sample, and finally purification of AMP
samples contaminated by either non-specific analytes or buffer
salts is demonstrated.
[0126] Aptamers include oligonucleotides (e.g., 25-100 bases long)
that recognize a broad class of analytes, such as small molecules,
peptides, amino acids, proteins, viruses, and bacteria, via
specific affinity interaction. They can be derived from ribonucleic
(RNA) or deoxyribonucleic (DNA) acids, aptamers can be isolated
through an in vitro procedure called systematic evolution of
ligands by exponential enrichment (SELEX), whereby large
populations of random sequence oligomers (DNA or RNA libraries) are
continuously screened against a target molecule until highly
selective candidates are isolated and subsequently amplified.
Aptamers have been used in applications such as target validation,
drug discovery, diagnostics, therapy, and in particular, analyte
purification. Employed in the described microfluidic device,
aptamers allow specific extraction and thermally induced recovery
of biomolecular analytes.
[0127] The microfluidic device of the described embodiments
includes a microchamber packed with aptamer-immobilized microbeads
for biomolecule analyte purification, a microheater and temperature
sensor 2110 for thermally induced analyte release, and
microchannels equipped with a passive valve using surface tension
for transferring released analytes to a spotting outlet 2112
coupled to a MALDI analysis plate (FIG. 21). Samples and reagents
are introduced via the sample inlet 2111, whereas the bead inlet
2113 is provisionally used for packing the microchamber with
microbeads and is sealed afterward. The microchamber (3 mm.times.3
mmx 180 .mu.m) is tapered and includes structural weirs to retain
the microbead support matrix on which the aptamer is immobilized.
Such a design minimizes dead volume and bubble formation in the
device. Hence, the chamber has an effective volume of .about.1.62
.mu.l with the tapers taken into consideration. For thermally
induced reversible disruption of aptamer-analyte binding, a
serpentine resistive heater and temperature sensor are placed below
the aptamer chamber 2114 to promote efficient heating of the entire
chamber and accurate sensing at the center of the microchamber.
Using Cr/Au thin films, a 500.OMEGA. heater and 27.5.OMEGA.
temperature sensor (with a temperature coefficient of resistance of
2.65.times.10.sup.-3/.degree. C.) can be used in conjunction with
off-chip programming to control temperature and thus, vary thermal
stimulation. The valve and deposition outlet are placed near the
aptamer chamber to reduce analyte dilution after release due to
adsorption to the channel walls or diffusion into dead fluid
volumes. The device also includes a waste reservoir 2115.
[0128] The microfluidic chip structure is realized with three
sandwiched polymer layers 2120-2122 (FIG. 21b). The bottom layer
2122 incorporates the inlets, passive valve 2132, and waste outlet
2134. To reduce bubble entrapment or dead volumes during sample
spotting, the middle layer 2121 provides an air vent 2126 connected
to the spotting outlet 2142. This provides a means for denser
fluids to force trapped gas in the spotting outlet out through the
air vent. Additionally, the middle layer 2121 encapsulates and
seals the microfluidic network formed in the bottom layer 2122.
Lastly, the top layer 2120 defines the spotting outlet 2152 and
houses the air vent channel 2154. To interface the device to the
MALDI, the device incorporates a glass capillary which is fitted to
the opening of the microchip spotting outlet. For example, samples
are infused from the capillary tip by hydrodynamic force and
allowed to crystallize before mass spectrometry analysis.
[0129] During operation, an aqueous sample containing a
biomolecular analyte intermixed with non-target molecules is
introduced to the aptamer-functionalized surfaces within the
microchamber, and thus is extracted by the aptamer (FIG. 21a). This
occurs at a suitable (e.g., room) temperature so that the aptamer
specifically captures the analyte from the liquid-phase while
impurities are flushed from the apparatus through the waste outlet.
The above sequence is repeated in a discrete (consecutive infusion
of dilute sample) fashion in order to adequately purify and enrich
the analyte, if necessary. Subsequently, the aptamer interaction
mechanisms can be disrupted by altering the temperature of the
solid support, such that the concentrated analyte is released into
a plug of pure aqueous buffer, or MALDI matrix solution. Thus, the
analyte can be isocratically eluted onto a MALDI analysis plate for
MS detection. Returning the temperature to the initial state allows
the aptamer to revert to its initial functional structure, i.e.,
the aptamer-functionalized surfaces are regenerated.
[0130] A microvalve is used to direct the purified analyte through
a secondary channel to the spotting outlet, and deposited onto the
MALDI analysis plate for MS detection (FIG. 21b). The microvalve
(FIG. 21a) exploits surface tension in that a pressure difference
exists at the air-liquid interface in a sudden restriction of a
hydrophobic channel. This pressure difference serves as a pressure
barrier: if and only if the pressure drop between the sample inlet
to the valve exceeds the pressure barrier, the eluent will enter
the secondary channel leading to the spotting outlet. This pressure
drop is primarily determined by the microchamber, whose flow
resistance is mainly due to the packed microbeads.
[0131] To demonstrate the functionality of the device, a model
binding apparatus is used which includes AMP analyte which is
recognized by an RNA aptamer derived for adenosine triphosphate
(ATP-aptamer). AMP is captured by ATP-aptamer through an induced
11-base loop flanked by double-stranded RNA, which forms an
affinity binding epitope for the small molecule.
[0132] Adenosine triphosphate aptamer (ATP-aptamer), with a 5'-end
functionalized with biotin, is acquired. As MALDI-MS can be
sensitive to salt impurities, DNA grade water (sterile
RNase/Protease-free water) is used to prepare ATP-aptamer, AMP,
cytidine, uridine, and guanosine triphosphate (CTP, UTP, and GTP,
respectively) samples in addition to being used for all washes. An
aqueous buffer solution (e.g., pH 7.4) is prepared with a mixture
of water, Tris-HCl (20 mM), NaCl (140 mM), KCl (5 mM), and
MgCl.sub.2 (5 mM). The MALDI matrix solution (THAP) is prepared
from 2,4,6-trihydroxy-acetophenone (2,4,6-THAP), 2,3,4-THAP, and
diammonium citrate at 0.1, 0.05, and 0.075 M concentrations,
respectively in a 3:5 (v:v) mixture of acetonitrile/water.
UltraLink streptavidin coated bis-acrylamide/azlactone beads (e.g.,
50-80 .mu.m in diameter) are used to immobilize ATP-aptamer via a
biotin-streptavidin link. Microfabrication materials, including
SU-8 2025 and 2100, Remover PG, Sylgard 184 polydimethylsiloxane
(PDMS), Torr Seal epoxy, polyethylene (PE) film, and microscope
grade glass slides (25 mm.times.75 mm), are used, respectively. A
DC power supply and a proportional-integral-derivative (PID)
controlled LabVIEW program are used in parallel to control
temperature during thermally-initiated analyte release from aptamer
molecules. Lastly, a syringe pump is used for sample and
introduction while a time of flight mass spectrometer is used for
mass analysis.
[0133] The device of the described embodiment is fabricated from
PDMS and bonded on a glass substrate using standard soft
lithography techniques (FIG. 22). SU-8 masters for each
microfluidic layer are first generated on silicon wafers. PDMS
pre-polymer solution is mixed (e.g., 10:1/v:v) and then poured onto
individual masters. A PE film (e.g., coated with an adhesive on one
side) is subsequently layered over the prepolymer mixture, allowing
surface tension forces to make intimate contact between the
prepolymer and PE film. The master/prepolymer/transparency stack is
then clamped within a through-hole PDMS sandwich assembly and cured
for 45-60 min at 60.degree. C. This produces thin PDMS microfluidic
layers which can be peeled off from the masters via the PE films
for bonding to the glass substrate. Meanwhile, glass substrates are
diced (25 mm.times.30 mm) and drilled to create the inlets and
outlets. Subsequently, Cr/Au (5/100 nm) thin films are deposited
and patterned on the substrates via thermal evaporation and then
passivated with SiO.sub.2 using plasma-enhanced chemical vapor
deposition (PECVD), realizing the microheater and temperature
sensor. Following plasma (O.sub.2) treatment of each bonding
interface, all three PDMS layers and the glass substrate are
aligned and permanently bonded. A glass capillary tube 2201 (5
mm.times.0.5 mm I.D.) is then inserted into the spotting outlet and
fastened with Torr Seal 2202. Finally, microbeads 2203 are packed
into the microchamber and the fluidic ports are sealed.
[0134] The device is first primed with a water wash (10 .mu.l/min,
10 min). The following washing and loading schemes are identical.
These parameters are specified based on the microchamber size (1.62
.mu.l), which is created such that with the microbeads packed at an
expected 63.5% efficiency, a fluid volume slightly over 1 .mu.l
(1.02 .mu.l) can be attained. This is within the common volume
range for sample spotting used in MALDI-MS analysis (0.5-2 .mu.l).
Initially, a 10 .mu.M ATP-aptamer sample (10 .mu.l) is loaded into
the microchamber and allowed to incubate with the streptavidin
functionalized bead bed (40 min). After subsequent washing, the
device is primed. In parallel, a device not functionalized with
ATP-aptamer (control device) is used to process control samples of
AMP, CTP, UTP, GTP (1 .mu.l; similar for all sample/matrix volumes
in the following demonstrations) which are prepared at 1 .mu.M
each. The operational principle described above is used. Manually
pipetted AMP, CTP, UTP, and GTP samples at 1 .mu.M concentrations
are deposited and analyzed to obtain reference spectrums. The
separate data sets are compared to reveal sample loss incurred
within the device during device operation.
[0135] For extraction and purification demonstrations using the
microdevice, 10 nM, 100 nM and 1 .mu.M AMP samples are loaded into
the aptamer microchamber separately. A rinse follows to rid
non-specific compounds. AMP is then released from the aptamer by
raising the chamber temperature to 45.degree. C. while introducing
a matrix sample plug. The sample/matrix plug is then transferred to
the spotting outlet and deposited onto the MALDI plate to be
subsequently analyzed. Similarly, for applications concerning
specific purification of AMP from model impurities, a solution of
AMP (100 nM), CTP (1 .mu.M), UTP (1 .mu.M) and GTP (1 .mu.M) is
loaded into the microchamber. After incubation (e.g., 3 min), the
impurity molecules are washed from the microchamber and matrix is
introduced. Heat is applied, while the passive valve activated, to
release the molecules currently on the aptamer and deposit them
onto the MALDI plate for analysis.
[0136] For enrichment and enhanced detection of AMP, a multiple
infusion scheme is used. The aptamer chamber is consecutively
loaded with 10 nM infusions of AMP sample. Each infusion is
incubated (3 min) and followed by a rinse. Upon suspected
saturation of the aptamer with AMP, the microchamber is heated to
release the analytes into a matrix plug, which is deposited for
analysis.
[0137] To ensure the validity of higher-level data, properties of
the valve in addition to absorption/adsorption characteristics of
the microfluidic structure are obtained. At a steady flow rate, the
pressure difference imparted by the passive valve impinges flow to
access the spotting outlet. When the waste outlet is open and the
flowrates are below 50 .mu.l/min (e.g., 10 .mu.l/min), fluid flow
bypasses the passive valve since the hydrodynamic pressure driving
flow (.about.686 Pa) is smaller than the actuation pressure of the
valve. To direct flow to the MALDI plate, the pressure drop between
the sample inlets to the waste outlet can be greater than the
valve's actuation pressure (i.e., above 3.154 kPa). This can be
accomplished by plugging the waste outlet using an external valve
and maintaining a constant flow rate during analyte sample
deposition following thermally induced release of biomolecules from
the aptamer. Additionally, analyte loss during fluidic transfer
from the microchamber to the spotting outlet is likely negligible,
since data obtained from samples spotted using the control device
match consistently to reference samples which are manually pipette
and deposited onto the analysis plate.
[0138] To demonstrate the ability to extract and detect AMP by
MALDI-MS using the device of the described embodiment, discrete
samples of varying concentration of AMP (10 nM, 100 nM and 1.0
.mu.M) are infused into the chamber. After interaction with the
aptamer functionalized beads, the AMP molecules are released and
transferred to the spotting outlet and finally deposited onto a
MALDI-MS plate. Mass analysis follows (FIG. 23). For attempted
extraction and detection of samples with concentration at 10 nM
(FIG. 23a), little or no signal can be obtained above the noise
level. In fact, the present mass peaks corresponding to THAP matrix
are limited to 339.44, 392.45, 468.23 and 502.05 Da/z. However, the
mass spectrum of a spot obtained from a 100 nM AMP solution (FIG.
23b) shows a distinctive mass peak of 348.11 Da/z, which
corresponds to AMP (established value: 347.22 Da/z) and indicates
that the potential detection range of the device of the described
embodiment lies between 10-100 nM. Since AMP concentration is still
relatively low for this case, the magnitude of its peak is
comparable to several peaks from the MALDI matrix (393.99 and
468.65 Da/z). Nonetheless, this detection sensitivity is c.a. one
order lower than physiologically relevant AMP levels in plasma. In
addition, a mass spectrum obtained from a 1.0 .mu.M AMP solution
(FIG. 23c) improves the analyte-to-matrix peak contrast. In this
case, the AMP peak dominates matrix peak amplitudes and indicates a
nonlinear dependence of detection signal to infused sample
concentration. Furthermore, this suggests that concentrating
undetectable dilute samples can improve the analyte detection
limit.
[0139] In another embodiment, for high sensitivity MALDI-MS,
analyte sample conditioning and enrichment can be useful to improve
the detection signal. The device of the described embodiment can be
used to enhance a sample of AMP (10 nM), previously established
undetectable, by loading the dilute AMP sample into the aptamer
chamber multiple times to saturate the analyte on the aptamer
before release and mass spectrometric analysis. A dilute sample
concentration is chosen to be much lower than 100 nM in order to
highlight the detection enhancement due to this technique of
enrichment. 25 consecutive dilute AMP samples are infused into the
aptamer chamber, the captured AMP are released with heat into a
pure matrix solution, and the concentrated plug is transferred to
the spotting outlet. A spectrum is obtained from the resulting
sample spot (FIG. 24a). An AMP peak to reference matrix ratio is
observed which is slightly higher than that seen in FIG. 23b,
demonstrating the successful concentration of AMP by
.about.10.times.. This result demonstrates the effectiveness of the
microchip for enhancing the detection of low concentration analytes
so as to facilitate label-free detection by MALDI-MS.
[0140] In yet another embodiment, to emphasize the capacity of this
approach, more consecutive infusions of dilute (10 nM) AMP solution
are performed in order to achieve a maximum enrichment factor for
the device of the described embodiment. A maximum of 250 infusions
are performed (FIG. 24b). Following the final infusion, a sample
spot is obtained and analyzed with MALDI-MS similar to the protocol
above with 25 infusions. Note that the AMP peak dominates those of
reference peaks and the AMP peak to noise peak ratio is comparable
to that of FIG. 23c. This suggests an AMP analyte enrichment factor
of nearly 100.times.. This is a substantial concentration factor,
comparable to that seen in reverse-phase SPE devices, but with the
benefit of higher specificity and affinity imparted by aptamers.
Moreover, by using the described enrichment protocol, the detection
limit of the device to AMP is improved by an order of magnitude and
now allows AMP detection at concentrations two orders below
physiologically relevant levels. In some embodiments, AMP sample
infusions are stopped after 250 since satisfactory signal
enhancement is achieved at this time, not because of actual
saturation of the analyte on the aptamer microbeads. The signal
gain achieved in FIG. 24b is merely the apparent signal
enhancement, since the potential for even larger enrichment factors
and higher signal gain is possible with the microchip. The
microchip can be regenerated (e.g., using thermal stimulation of
the aptamer functionalized beads) to allow reuse and repeated
functionality.
[0141] Purification of analytes can be a useful tool for
selectively controlling analytes in biochemical applications. In
other embodiments, the signal of AMP (100 nM) can be selectively
isolated and enhanced from a homogeneous solution amongst CTP, UTP,
and GTP (model nonspecific analytes at 1.0 .mu.M each) by loading
the sample into the microchamber and subsequently washing the
chamber to isolate AMP on the aptamer functionalized beads. To
emphasize the power of aptamer purification, the ratio of AMP to
non-specific impurity analytes is reduced (1:10) in order to mimic
more closely a common practical situation in which a target analyte
can be in unfavorable disproportion to non-target analytes. A
deposited sample spot is obtained similarly to previous protocols.
The control device is used to establish a reference spectrum for an
unclean sample. Both samples are compared to delineate the
effectiveness of aptamer based sample cleanup prior to MALDI-MS
(FIG. 25). For the control sample (FIG. 25a), the ratio of AMP to
matrix is comparable to that seen in FIG. 23b, where only AMP is
present in the solution. However, the non-target peaks
corresponding to the model impurities are observed: CTP (480.01),
UTP (484.51), and GTP (523.74) Da/z which can have an adverse
effect on the signal quality. This is unlike the signal obtained
utilizing the aptamer functionalized microchip, where cleaning of
the AMP sample through extraction and purification is possible
(FIG. 25b). A reduction of impurity peaks (e.g., that of CTP)
exists, at the same time that the AMP signal is improved. Although
the CTP, UTP and GTP are still present, their intensities are lower
than the AMP peak for this case, suggesting that the amount of
non-specific binding is negligible to the AMP-specific aptamer.
Non-specific binding can degrade MALDI-MS detection for practical
applications.
[0142] Along with potential interference from non-specific
analytes, MALDI analysis can also be hindered by contamination of
salts present in both conditioned solutions and physiological
solutions. Since a particular analyte can be solvated within a
solution stemming from one of these sources, addressing this type
of contamination in analytical samples is useful before performing
MALDI-MS. the microdevice is capable of selectively isolating AMP
and enhancing its detection from a buffer solution contaminated
with common pH altering salts (e.g., Tris-HCl, NaCl, KCl, and
MgCl.sub.2). These compounds can degrade the baseline generated for
a given MALDI spectrum (e.g., the baseline is translated
considerably above 0 Da/z), which can alter the relative
intensities of significant analyte peaks as well as produce
unwanted noise. A 100 nM AMP sample in buffer solution is initially
desalinated by infusing the sample into the aptamer microchamber to
allow specific interaction of the AMP to ATP-aptamer. Flushing the
buffer solution through the waste outlet followed by a short wash
procedure allows the analyte to be purified. This is followed with
an infusion of a pure matrix plug and simultaneously initiating
thermal release, sample transfer to the spotting outlet, and
deposition of the analyte similar to previous protocols. The
control microchip is used similarly to that described above to
establish a reference spectrum for the salt laden sample. The
spectrums are compared to reveal the effective desalting capability
of the device (FIG. 26). The control sample spectrum (FIG. 26a)
reveals characteristic properties of a salt contaminated sample.
For example, the baseline of the spectrum is raised significantly
above 0 Da/z, altering the relative ratios of significant mass
peaks. The AMP mass peak is barely registered above the baseline
and noise peaks (265.90 Da/z), due to buffer salts, dominate
instead. After using the aptamer functionalized chip for the same
AMP sample, a reduction of the baseline to near 0 Da/z in can be
observed addition to an enhanced AMP mass peak signal (FIG. 26b).
There is a reduction of all impurity and salt peaks (e.g. 265
Da/z), which highlights the benefits of this microchip for
desalination sample conditioning before MALDI-MS.
[0143] Other embodiments illustrate a microfluidic approach to
characterizing biomolecular binding properties. The techniques
include a microfabricated chip with biomolecule functionalized
surfaces coupled to a matrix-assisted laser desorption/ionization
mass spectrometer (MALDI-MS). The thermally-dependent binding
properties of adenosine monophosphate, vasopressin, and
platelet-derived growth factor can be observed with their
respective aptamer receptors. A binding profile for each
biomolecular pair revealed zones of either strong or weak
interaction depending on the localized temperature. This platform
can be useful for screening therapeutic and receptor ligands.
[0144] Some embodiments illustrate a label-free, microfluidic
approach to characterizing the temperature-dependant nature of
receptor-analyte interactions. The techniques are demonstrated with
three devices based on synthetic affinity oligonucleotide
receptors, aptamers and their specific analytes: (1) adenosine
monophosphate (AMP) with an anti-AMP RNA aptamer; (2) platelet
derived growth factor (PDGF) and its specific DNA aptamer; and (3)
vasopressin with a specific RNA aptamer (called a spiegelmer). This
is accomplished using an integrated microfluidic device coupled to
label-free detection with matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-MS).
[0145] To characterize temperature dependent receptor-ligand
binding, the receptor is immobilized on solid surfaces (FIG. 27).
At each of a selected series of temperatures, a sample containing
the ligand, with a non-binding species for use as a reference
standard in the subsequent quantification stage, is introduced in a
defined volume to the receptor-functionalized solid surfaces (FIG.
27a). Alternatively, the ligand can be surface-immobilized while
the receptor is solution-borne in the sample. The sample is then
incubated for a sufficient time period, so that some of the ligand
molecules bind with the receptor, while the reference standard
remains in solution (FIG. 27b). The sample, now containing the
ligand molecules not bound to the receptor along with the reference
standard molecules, is transferred to a MALDI-MS analysis plate,
and subsequently quantified by mass spectrometry (FIG. 27e). The
normalized mass spectral peak of the ligand, defined as the ratio
of the mass spectral peaks of the ligand and reference standard,
will vary at the different temperatures. This relationship
represents the temperature dependence of the equilibrium binding
between the ligand and receptor. Also, initially bound molecules
can be released by modifying the surface temperature above or below
a binding temperature (FIG. 27c, d), while introducing a
non-binding reference sample, to illustrate the efficiency of
temperature dependent dissociation.
[0146] Using this approach, the temperature-dependent interaction
properties between AMP and its specific aptamer is illustrated.
However, in this apparatus, an anti-AMP aptamer is immobilized on
microbeads while AMP in solution is introduced for binding,
release, and subsequent MALDI-MS detection. Guanosine monophosphate
(GMP) is utilized as a standard non-binding analyte. Using a single
concentration of 10 .mu.M AMP, binding is characterized from room
temperature to 60.degree. C. (FIG. 29). The AMP apparatus
demonstrates optimal binding within the temperature range of
25-35.degree. C., indicated by the low AMP to GMP mass peak ratio.
Binding dissociation initiates in a temperature zone immediately
higher than 35.degree. C. and increases above 45.degree. C.
[0147] Following the same protocol as AMP and its specific aptamer,
the temperature dependent binding characteristics of PDGF and its
correlating specific aptamer are illustrated. However, in this
device, a PDGF specific aptamer is immobilized on microbeads while
PDGF in solution is introduced for binding, release, and subsequent
MALDI-MS detection. Furthermore, vascular endothelial growth factor
(VEGF) is used as a non-binding standard. Using a single
concentration of 10 .mu.M PDGF, binding is characterized from room
temperature to 60.degree. C. (FIG. 30). In this case, good binding
is illustrated in one temperature zone, 24-45.degree. C. as
indicated by a low normalized PDGF/standard peak. Release of PDGF
from its specific DNA aptamer occurred in a single observable
temperature zone: 45-60.degree. C.
[0148] The polypeptide, vasopressin, from room temperature to
75.degree. C. is then characterized (FIG. 31). A wide spiegelmer
concentration range (0.01, 0.1, 1 and 10 .mu.M) with equal
concentrations of P18 standard was used. For example, with a 10
.mu.M spiegelmer/standard sample, good binding is illustrated in
two temperature zones, 34-45.degree. C. and 70-75.degree. C. This
is indicated by a low normalized spiegelmer/standard peak. Release
of spiegelmer from the vasopressin (indicated by a high normalized
peak) occurs in three temperature zones: 15-30.degree. C.;
50-65.degree. C.; and above 75.degree. C. This is similar for all
demonstrated concentrations, indicating consistency over three
orders of magnitude.
[0149] Thus, the described apparatus can be used as a powerful tool
for label-free characterization of temperature dependent binding of
biomolecular targets with aptamers. Such complex binding profiles
can be difficult to elucidate with conventional approaches.
Additionally, these concepts provide techniques for surface-based
biosensor characterization.
[0150] Further embodiments illustrate a microfluidic aptameric
biosensor, or aptasensor, for selective detection of clinically
relevant analytes with integrated analyte enrichment, isocratic
elution and label-free detection by mass spectrometry. Using a
microfluidic platform that is coupled to matrix assisted laser
desorption/ionization mass spectrometry (MALDI-MS), specific
purification, enrichment, and label-free detection of trace amounts
of arginine vasopressin (AVP), a peptide hormone that is
responsible for arterial vasoconstriction is demonstrated. During
extreme physical trauma, in particular immunological shock or
congestive heart failure, AVP is excreted abnormally and is hence a
biomarker for such conditions. The device uses an aptamer, e.g., an
oligonucleotide that binds specifically to an analyte via affinity
interactions, to achieve highly selective analyte capture and
enrichment. In addition, via thermally induced reversible
disruption of the aptamer-analyte binding, the device can be easily
regenerated for reuse and allows isocratic analyte elution, i.e.,
release and collection of analytes using a single aqueous
solution.
[0151] Furthermore, the device of the current embodiment is coupled
to MALDI-MS using a microfluidic flow gate, which directs the
eluted analyte onto a MALDI sample plate for mass spectrometry.
First, systematic characterization of kinetic and thermal release
properties, as well as the overall timescale of the assay, is
performed using fluorescently labeled AVP. Then, MALDI-MS detection
of unlabeled AVP at clinically relevant concentrations approaching
1 pM is illustrated.
[0152] In the current embodiment, the specific aptameric isolation
and enrichment, as well as label-free MALDI-MS detection, of AVP is
illustrated. Using a microfluidic device of the present
embodiments, AVP is first selectively captured and enriched on an
aptamer-functionalized solid phase, and then collected using
microflow gating and thermally induced isocratic elution (i.e.,
elution within the same aqueous phase for analyte capture) on a
MALDI sample plate for mass spectrometric analysis. Systematic
characterization of the aptasensor is first illustrated using
fluorescently labeled AVP, including time-resolved measurements of
aptamer-AVP interaction, concentration-dependent fluorescence
response of AVP, temperature-dependent aptamer-AVP dissociation,
and detection of trace AVP after enrichment of a dilute sample.
Label-free detection of AVP, in the presence of significant levels
of model impurities, at both physiologically critical
concentrations (i.e., during symptoms of immunological shock and
renal congestive heart failure) and normal conditions is then
illustrated. In this way, the required time to detect AVP is
reduced to within a working day (compared to 3-11 days for
conventional approaches), while eliminating unnecessary chemical
modification protocols such as those required for fluorescent or
radiometric probes. Moreover, by integrating MEMS technology with
microfluidics, these techniques provide a foundation for
point-of-care and automated diagnosis of AVP maladies.
[0153] OF THE DESCRIBED EMBODIMENT includes a microchamber 3201
packed with aptamer-functionalized microbeads for sample
purification, a microheater and temperature sensor 3202 for
thermally induced analyte release, and microchannels equipped with
a surface tension-based passive microflow gate and air vent for
transferring released sample to a spotting outlet 3203 coupled to a
MALDI sample plate (FIG. 32a). FIG. 32a also shows a sample inlet
3204, a bead inlet 3205, a valve 3206 and a waste outlet 3207.
Structurally, the device includes a glass coverslip bonded to three
stacked polymer layers (FIG. 32b): the bottom layer 3210
incorporates the inlets 3220, passive flow gate 3222, and waste
outlet 3224; the middle layer 3211 contains the air vent 3226 and
seals the bottom-layer microfluidic features; and the top layer
3212 defines the spotting outlet 3232, to which a glass capillary
3228 is fitted to allow sample ejection to a MALDI sample plate.
The top layer 3212 also houses the air vent channel 3230, whose
hydrophobic surface allows trapped gas bubbles to be eliminated
from the spotting outlet 3232. Microbeads on which aptamer
molecules are immobilized are packed inside the microchamber
(volume: 1.6 .mu.m) and retained by dam-like micro weirs. A
thin-film resistive metal heater and temperature sensor are
integrated on the glass surface to allow on-chip, closed-loop
temperature control. FIG. 32c shows another view of the spotting
outlet and the aptamer chamber 3208.
[0154] During operation, an aqueous sample containing AVP
potentially intermixed with non-target molecules is introduced into
the aptamer microchamber, and thus is extracted by the aptamer.
This occurs at a suitable temperature (e.g., .about.37.degree.C.)
so that the aptamer specifically captures the target from the
liquid-phase while impurities are flushed from the apparatus
through the waste outlet. The above sequence is repeated in a
continuous fashion in order to adequately purify and enrich the
analyte, if necessary. For MALDI-MS analysis, the aptamer
interaction mechanisms can be disrupted by altering the temperature
of the solid support, such that the concentrated analyte is
released into a plug of pure MALDI matrix solution. Subsequently,
the microflow gate is utilized to transfer the plug to the spotting
outlet by exploiting the pressure difference induced across an
air-liquid interface in a hydrophobic channel restriction. If the
pressure drop between the sample inlet to the flow gate exceeds
this pressure difference, fluid can enter the secondary channel
leading to the spotting outlet. Hence, the fluid can be switched
between the channels that access the spotting outlet or bypass it
to the waste outlet. The air vent connected to the spotting outlet
provides a means to reduce bubble entrapment or dead volumes during
sample spotting. Thus, purified and enriched samples are ejected
from the capillary tip by hydrodynamic force and allowed to
crystallize before mass spectrometry analysis. This preceding
protocol allows isocratic elution of analytes onto a MALDI sample
plate for MS detection. Returning the temperature to the initial
state allows the aptamer to revert to its initial functional
structure, i.e., the aptamer-functionalized surfaces are
regenerated.
[0155] The device of the present embodiment can be fabricated by
standard soft lithography techniques. Briefly, sheets of
polydimethylsiloxane (PDMS) bearing the microfluidic features are
obtained by micromolding using a master fabricated from SU-8 on
silicon, while the microheater and temperature sensor are
fabricated from a 100 nm gold thin film (using a 5 nm chromium
adhesion layer) on glass. Each PDMS sheet is then bonded to the
glass chip, as shown in FIG. 32b, after treating the bonding
interfaces by oxygen plasma. Microfabrication materials, for
example, SU-8 2025 and 2100 (MicroChem), Sylgard 184 PDMS, Ton Seal
epoxy, and microscope grade glass slides (25 mm.times.75 mm), are
obtained.
[0156] Illustration of the described techniques involves systematic
device characterization using fluorescently labeled AVP with
respect to adenosine monophosphate (AMP) as a model impurity, and
demonstration of capture, enrichment and MALDI-MS detection of
unlabeled AVP from AMP. The AVP-specific aptamer, termed a
spiegelmer, is derived from an L-type enantiomeric RNA sequence
(5'-Biotin-(HEG18) GGGGUAGGGCUUGGAUGGGUAGUACAC (HEG18)
GUGUGCGUGGU-3', HEG18 is a hexaethylene glycol linker) and this
sequence resists degradation by free ribonucleases. It is
synthesized using nuclease-resistant L-type enantiomeric
nucleotides. Meanwhile, unlabeled AVP is synthesized on peptide
synthesizer, and AVP labeled with the fluorescent dye Tamra
(TMR-AVP) is synthesized by coupling AVP peptide with dye Tamra.
Unlabeled AMP is obtained while AMP labeled with the fluorescent
dye thiazole orange (TO-AMP) is synthesized by coupling
TO-hydroxysuccinimidyl ester with AMP-NH-linker-NH.sub.2.
Analytical samples used during fluorescently based characterization
experiments involving TMR-AVP are prepared in buffer solution
(AVP-buffer, pH 7.4) including purified water (sterile
RNase/Protease-free), Tris-HCl (20 mM), NaCl (150 mM), KCl (5 mM),
CaCl.sub.2 (1 mM), and MgCl.sub.2 (1 mM); while samples utilized in
MALDI-MS protocols required solvation in purified water. A MALDI
matrix, cyano-4-hydroxycinnamic acid is solvated in a volume ratio
mixture of 50:50:0.3 purified water/acetonitrile/trifluoroacetic
acid. Porous bis-acrylamide beads copolymerized with azlactone
(50-80 .mu.m in diameter) and coated with UltraLink streptavidin
are used to immobilize receptor or ligand moieties via a
biotin-streptavidin link.
[0157] The described devices are initially rinsed thoroughly (flow
rate: 10 .mu.l/min) with purified water for 30 minutes (similar for
subsequent rinses in all experiments). Sample solutions in varying
concentrations of TMR-AVP and unlabeled AVP are prepared using the
appropriate mass weights of the respective compound and either
AVP-buffer (for TMR-AVP), or water (for AVP) solution. Manual
pressure was utilized to pack microbeads from the bead introduction
channel of the aptasensor into the microchamber. After another
rinse procedure, an AVP-aptamer solution (50 .mu.M) was injected (3
.mu.l, 50 .mu.l/min) and allowed to incubate (40 min) in the
chamber. (This procedure was used for all sample injections.) A
inverted epi-fluoresence microscope (e.g., Nikon Eclipse TE300) is
used for fluorescence characterization. Initially, a baseline
fluorescence signal was acquired by focusing a 10.times. objective
at a central location in the extraction chamber and averaging an
8-bit RGB signal over the entire recorded fluorescence image.
Alternatively, MALDI-MS experiments are performed using a time of
flight mass spectrometer (e.g., from Applied Biosystems,
Voyager-DE).
[0158] Systematic characterization of the AVP-aptamer binding using
TMR-AVP (peak absorption: 540 nm; peak emission: 580 nm) is
performed. Then, MALDI-MS analysis is performed. The fluorescently
based characterization allows for the visualization the binding
characteristics of the apparatus.
[0159] Several initial procedures are performed using a
microchamber with and without AVP-aptamer functionalized beads. The
beads are introduced to samples of TMR-AVP and a model impurity,
TO-AMP (peak absorption: 480 nm; peak emission: 530 nm). After a
baseline fluorescence signal is acquired, the chamber is initially
packed with non-functionalized beads (streptavidin-coated
microbeads: ("bare beads")). Subsequently, samples of TMR-AVP and
TO-AMP (1 .mu.M) are injected into the microchamber. The resulting
fluorescence gain is measured. Similarly, TMR-AVP and TO-AMP
samples (1 .mu.M) are exposed to a chamber packed with AVP-aptamer
functionalized microbeads. As shown in FIG. 33, there is little or
no appreciable signal above the baseline in the bare beads case for
both TMR-AVP and TO-AMP samples, while merely an increase of 1.45%
in fluorescence over the baseline occurs when TO-AMP is introduced
to AVP-aptamer. In contrast, the fluorescent intensity from
introducing TMR-AVP to AVP-aptamer is dominant. TMR-AVP indeed
interacts with AVP-aptamer. Moreover, this result highlights the
specificity between the binding of AVP-aptamer and AVP.
[0160] The time course of affinity capture of TMR-AVP by the
aptamer is obtained to illustrate the kinetic behavior of the
apparatus. This is accomplished by recording the time-resolved
fluorescence response after introducing a TMR-AVP sample into the
aptamer microchamber. Fluorescence micrographs are taken at
discrete time intervals (5 s) following an injection of TMR-AVP in
varying concentrations (0.01, 0.1, and 1 .mu.M). To reduce the
effect of fluorescent photobleaching, the shutter to the mercury
lamp is closed for the time period between all signal measurements.
Fluorescence signal measurements are obtained, averaged and then
plotted as a function of time (FIG. 34). The fluorescence intensity
increases steadily with time until sufficient signal saturation
occurs. The apparent AVP capture time (i.e., time constant for the
observed time course of AVP capture) is approximately 8.4, 13.5,
and 22.1 s for 1, 0.1, and 0.01 .mu.M TMR-samples, respectively.
The binding time can be affected by three time scales: thermal,
diffusion and kinetic. For the porous microbead-packed
microchamber, the diffusion time scale is approximately d.sup.2/D
.about.5.85 s, where d is the average bead diameter (50 .mu.m), and
D the analyte diffusivity (approximately .about.4.times.10.sup.-6
cm.sup.2/s for AVP). This result is significant compared with the
apparent analyte capture times seen in FIG. 36. Thus, the
interaction between AVP and its aptamer in this situation can
depend on both kinetics and diffusion. Further, the longer apparent
capture times observed for lower AVP concentrations agrees with
monovalent binding theory. Illustrated below, these apparent
capture times provide a basis for choosing the sample incubation
time (for concentration dependent fluorescence response) or
infusion flow rate (for analyte enrichment). To confirm
aptamer-based capture of AVP within the aptasensor microchamber,
solutions of TMR-AVP at five different concentrations (0.001, 0.01,
0.1, 1, and 10 .mu.M) were injected into the microchamber. After
each sample introduction, fluorescence yield was quantified after
an initial 30 s incubation time to assure equilibrium sample
binding. Following the extraction of AVP, the microchamber was
washed with buffer to rid all non-specific compounds, un-reacted
molecules, and impurities. Results are presented in FIG. 35. It can
be seen that below 1 nM, no measurable signal above the baseline is
detected. Concentrations at and above 1 nM, however, are readily
detectable for the aptasensor with c.a. a signal-to-noise ratio of
3. Additionally, the concentration dependent fluorescence signal
produced through TMR-AVP capture appeared to be dose-responsive.
This was signified by the S-shape fit of the data using GraphPad
Prism 5 (GraphPad Software) software. These results suggested the
need for enhanced detection techniques, such as analyte enrichment,
in order to render the aptasensor clinically viable; in other
words, enable detection of AVP below 1 nM at physiologically and
clinically relevant levels (e.g., 1-500 pM).
[0161] To investigate detection enhancement of TMR-AVP, a
continuous-flow analyte enrichment scheme is used. A dilute
solution of TMR-AVP (100 pM) is continuously infused into the
microchamber until fluorescence saturation is observed. Taking into
consideration the required residence time determined above, the
sample flow rate is chosen to be 15 .mu.l/min (corresponding to a
sample residence time of 20 s in the microchamber) to insure
complete AVP interaction with the aptamer. Fluorescence signals are
obtained periodically (e.g., every 60 min) until approximately 480
min, when no significant increase in fluorescence was observed,
which suggested saturation (FIG. 36). This agrees with the expected
equilibrium condition that concentration of a molecular analyte
onto a receptor modified surface increases consistently with time
at a decreasing rate. Moreover, the observed fluorescence response
of the original 100 pM sample corresponds to the apparent
fluorescence response of a 0.1 .mu.M TMR-AVP sample, indicating
significant detection enhancement. Hence, using the analyte
enrichment feature of the aptasensor demonstrates clinical
potential for vasopressin diagnostics since shock and congestive
heart failure AVP signaling levels in plasma are c.a. 100-500 pM.
Moreover, the required processing time to perform enhanced
detection of TMR-AVP with the aptasensor, although seemingly long
(8 hrs), is drastically improved over conventional techniques,
which require nearly 11 days.
[0162] Characterization of the temperature-dependant reversibility
of AVP and AVP-aptamer binding enables label-free MALDI-MS
detection. This is accomplished in the device of the described
embodiments by thermally activated release and isocratic elution of
analytes from aptamer-functionalized microbeads. To demonstrate
this, a 1 .mu.M TMR-AVP solution is first introduced into the
microchamber and allowed to associate with AVP-aptamer. After
binding of TMR-AVP on the aptamer surface, a high intensity
fluorescence signal is observed (FIG. 37). The temperature on-chip
is then increased to a predetermined setpoint and held for 2 min
while AVP-buffer is flowed into the microchamber. The procedure is
repeated for several elevated setpoint temperatures ranging from
34-60.degree. C. A sharp decrease (93%) in fluorescence intensity
occurs at 50.degree. C. that continues until fully suppressed at
58.degree. C., indicating nearly complete reversal of TMR-AVP
binding on the microbeads. The effect of photobleaching is
determined negligible. This demonstrates the capability of the
aptasensor of the described embodiment for thermally activated
release and isocratic elution of a captured target. This technique
is also used to perform repeated demonstrations with TMR-AVP
samples within the same aptasensor chip. The fluorescence signals
resulting from TMR-AVP extraction and release in all demonstrations
produces consistent and repeatable values, as reflected by the
error bars on the data. This indicates that the thermal stimulation
does not affect the functionality of the aptamer molecules and
successfully allows for aptasensor regeneration and repeated
use.
[0163] To demonstrate the ability to extract and detect AVP by
MALDI-MS, discrete samples of physiologically relevant
concentrations of AVP (1 pM, 10 pM, 100 pM, and 1 nM) are first
introduced into the chamber. After interaction with the aptamer
functionalized beads, the AMP molecules are thermally released and
transferred to the spotting outlet via the passive microflow gate
and finally deposited onto a MALDI-MS plate. Mass analysis follows
(FIG. 38). No molecular ion peak registers for the 1 pM sample
(FIG. 38a). In fact, only mass peaks corresponding to the HCCA
matrix and its fragments/adducts are present (377.6, 648.2 and
860.5 Da/z). However, the mass spectra of a spots obtained for all
other AVP solutions (FIG. 38b-d) shows a distinctive molecular ion
peak of 1084.4 Da/z that corresponds to AVP. For example, sample
AVP concentrations between 10 pM and 1 nM (FIG. 38b-d) demonstrates
improved detection and signal-to-noise ratio with increasing
concentration. However, since AVP concentration is still rather low
for sample concentrations between 10 pM and 1 nM, the magnitude of
its peak is smaller than the MALDI matrix peaks (e.g., 377.6 Da/z).
Moreover, this detection sensitivity is c.a. on the order of
average physiological AVP levels in plasma. To improve detection at
low levels (e.g., 1-100 pM) analytes can also be enriched in the
aptasensor prior to MALDI-MS.
[0164] Naturally occurring hormonal vasopressin is present in
plasma predominantly above 1 pM. To demonstrate detection at this
level and therefore pervade all clinical settings, poorly, or
undetectable, samples of AVP are enriched using continuous infusion
of an original dilute AVP solution. Particular protocol parameters,
such as saturation time (.about.8 hrs) and flow rate (15 .mu.l/min)
are drawn from the above demonstrations. Following a similar
process, dilute samples of AVP (1, 10, and 100 .mu.M) are
continuously infused into the aptamer chamber for the designed time
period. This is followed by thermally induced release of the
captured AVP into a pure matrix solution (1 .mu.l), and subsequent
transfer of the enriched analyte plug to the spotting outlet. A
mass spectrum is obtained from the resulting sample spot (FIG. 39).
For each original dilute sample, there is an enhanced detection of
the molecular ion peak for AVP. Specifically, the original 1, 10
and 100 pM samples produce mass spectra where the AVP peak compares
to (or is better than, in the case of the 100 pM sample) 100 pM, 1
nM, and 100 nM samples measured in the previous demonstration,
respectively. Notably, the 1 pM sample, which was undetectable
before analyte enrichment, became quantifiable afterwards. Although
the repeated use of the aptasensor is not explicitly gleaned from
the presented data (due to the limits of presenting spectroscopic
data), the aptasensor is easily regenerated (using thermal
stimulation of the aptamer functionalized beads) to allow reuse and
repeated functionality.
[0165] The foregoing merely illustrates the principles of the
described subject matter. Various modifications and alterations to
the described embodiments will be apparent to those skilled in the
art in view of the teachings herein. It will thus be appreciated
that those skilled in the art will be able to devise numerous
techniques which, although not explicitly described herein, embody
the principles of the described subject matter and are thus within
its spirit and scope.
* * * * *