U.S. patent application number 14/175835 was filed with the patent office on 2015-10-01 for real-time, single-step bioassay using nanoplasmonic resonator with ultra-high sensitivity.
This patent application is currently assigned to The Regents of the University of California. The applicant listed for this patent is The Regents of the University of California. Invention is credited to Fanqing Frank CHEN, Jonathan A. ELLMAN, Kai-Hang SU, Cheng SUN, Qi-Huo WEI, Xiang ZHANG.
Application Number | 20150276610 14/175835 |
Document ID | / |
Family ID | 40901571 |
Filed Date | 2015-10-01 |
United States Patent
Application |
20150276610 |
Kind Code |
A1 |
ZHANG; Xiang ; et
al. |
October 1, 2015 |
REAL-TIME, SINGLE-STEP BIOASSAY USING NANOPLASMONIC RESONATOR WITH
ULTRA-HIGH SENSITIVITY
Abstract
A nanoplasmonic resonator (NPR) comprising a metallic nanodisk
with alternating shielding layer(s), having a tagged biomolecule
conjugated or tethered to the surface of the nanoplasmonic
resonator for highly sensitive measurement of enzymatic activity.
NPRs enhance Raman signals in a highly reproducible manner,
enabling fast detection of protease and enzyme activity, such as
Prostate Specific Antigen (paPSA), in real-time, at picomolar
sensitivity levels. Experiments on extracellular fluid (ECF) from
paPSA-positive cells demonstrate specific detection in a complex
bio-fluid background in real-time single-step detection in very
small sample volumes.
Inventors: |
ZHANG; Xiang; (Alamo,
CA) ; ELLMAN; Jonathan A.; (Guilford, CT) ;
CHEN; Fanqing Frank; (Moraga, CA) ; SU; Kai-Hang;
(Chungho, TW) ; WEI; Qi-Huo; (Hudson, OH) ;
SUN; Cheng; (Wilmette, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Assignee: |
The Regents of the University of
California
Oakland
CA
|
Family ID: |
40901571 |
Appl. No.: |
14/175835 |
Filed: |
February 7, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12772118 |
Apr 30, 2010 |
8685743 |
|
|
14175835 |
|
|
|
|
PCT/US2008/082266 |
Nov 3, 2008 |
|
|
|
12772118 |
|
|
|
|
60984859 |
Nov 2, 2007 |
|
|
|
Current U.S.
Class: |
506/10 ;
506/18 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 21/658 20130101; B82Y 20/00 20130101; C12Q 1/37 20130101 |
International
Class: |
G01N 21/65 20060101
G01N021/65; C12Q 1/37 20060101 C12Q001/37 |
Goverment Interests
STATEMENT OF GOVERNMENTAL SUPPORT
[0002] This work was supported by NSF Nano-scale Science and
Engineering Center (DMI-0327077), NSFSST/Collaborative Research
Program (DMI-0427679), and NASA Institute for Cell Mimetic Space
Exploration (CMISE), under Award No. NCC 2-1364; NHLBI/NIH HL078534
and NCl/NIH R1CA95393-01; DARPA, and UCSF Prostate Cancer SPORE
award (NIH Grant P50 CA89520; and P01 CA072006), under Contract no.
DE-ACO2-05CH11231 awarded by the U.S. Department of Energy, at the
University of California/Lawrence Berkeley National Laboratory.
Claims
1. A nanoplasmonic resonator (NPR) comprising a plurality of
metallic nanodisks with alternating shielding layer(s), having a
tagged biomolecule conjugated or tethered to the surface of the
nanoplasmonic resonator.
2. The resonator of claim 1 wherein the tag is a Raman active
tag.
3. The resonator of claim 1 wherein the tag is a fluorescent
tag.
4-5. (canceled)
6. The resonator of claim 1 wherein the shielding layer comprises a
material having a constant Raman spectra.
7. The resonator of claim 6 wherein the shielding layer is selected
from the group consisting of silicon dioxide, quartz, polystyrene,
silica, and dextran.
8. The resonator of claim 1 wherein the biomolecule is a peptide
linked to a Raman active tag, wherein the peptide comprising an
amino acid sequence that can be specifically modified or cleaved by
an enzyme.
9. (canceled)
10. A nanoplasmonic resonance SERS detection platform comprising a
patterned substrate featuring a surface enhanced Raman scattering
(SERS) nanoplasmonic resonator (NPR) singly or in an array, wherein
the nanoplasmonic resonator has a biomolecule conjugated
thereto.
11. The platform of claim 10 wherein the substrate on which the
nanoplasmonic resonator is patterned can be comprised of quartz,
polystyrene, silica, dextran, or any other materials with constant
Raman spectra.
12. The platform of claim 10 wherein the nanoplasmonic resonator
comprising metallic nanodisks with alternating shielding layer(s),
having a tagged biomolecule conjugated or tethered to the surface
of the nanoplasmonic resonator, wherein the tag is a Raman active
tag for SERS detection.
13. The platform of claim 12 wherein the nanodisk comprises a thin
layer of gold or silver.
14. The platform of claim 12 wherein the shielding layer comprises
silicon dioxide, quartz, polystyrene, silica, or dextran.
15. The platform of claim 10 wherein the biomolecule is a peptide
linked to a Raman active tag, wherein the peptide comprising a
specific sequence that can be specifically modified or cleaved by
an enzyme.
16. The platform of claim 10 wherein the nanoplasmonic resonators
in an array are the same or different.
17. The platform of claim 15, wherein a plurality of members of a
biomolecule library are conjugated to nanoplasmonic resonators
comprising said array and spatially separated in either a random
array or ordered microarray format.
18.-19. (canceled)
20. A method for in vitro detection and measurement of enzymatic
activity using a nanosensor comprising a nanoplasmonic resonator
(NPR) with at least picomolar sensitivity, wherein the NPR enhances
Raman spectra intensity in Surface-Enhanced Raman Spectroscopy
(SERS) and enables single-step detection of enzymatic activity.
21. A method for real-time reaction monitoring of enzyme activity
comprising: (a) providing an array of peptide-conjugated
nanoplasmonic resonators patterned on a substrate, wherein each
nanoplasmonic resonator (NPR) comprising metallic nanodisks with
alternating shielding layer(s) and having a tagged biomolecule
conjugated or tethered to the surface of the nanoplasmonic
resonator, wherein the tag is a Raman tag molecules, each being the
same or different, and wherein each peptide comprises a sequence
recognized and modified by a specific enzyme using a specific
reaction; (b) providing a biological sample suspected of containing
an enzyme, (c) contacting said sample with said array of
nanoplasmonic resonators; (d) allowing said reaction to be carried
out; and (e) measuring detection of the enzyme by SERS
detection.
22. The method of claim 21, wherein a biomolecule library is
conjugated to the nanoplasmonic resonators and spatially separated
in either a random array or ordered microarray format.
23. The method of claim 22, wherein the array of the
biomolecule-nanoplasmonic resonators detect the activity of
multiple enzymes simultaneously.
24-26. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/772,118, filed on Apr. 30, 2010, which is a continuation of
International Patent Application No. PCT/US2008/082266, filed on
Nov. 3, 2008, which claims priority to U.S. Provisional Patent
Application No. 60/984,859, filed on Nov. 2, 2007, all of which are
hereby incorporated by reference in their entirety. U.S.
application Ser. No. 12/772,118 also claims priority to U.S.
Provisional Patent Application No. 60/984,859, filed on Nov. 2,
2007.
REFERENCE TO SEQUENCE LISTING
[0003] The sequence listing in paper form is hereby incorporated by
reference in its entirety as attached to the specification.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention relates to the fields of Surface
Enhanced Raman Scattering (SERS) using a single and arrays of
nanoplasmonic resonators for detection of enzymatic activity. The
present invention relates specifically to the detection of protease
activity, such as the Prostate Specific Antigen (PSA) and
Proteolytically Active PSA for diagnostic applications in prostate
cancer.
[0006] 2. Related Art
[0007] Originally developed in 1928, Raman spectroscopy has been
used extensively to characterize molecular properties (Kazuo
Nakamoto John R. Ferraro, Chris W. Brown, Introductory Raman
Spectroscopy, 2nd ed. (Elsevier Science, 2003). Surface-Enhanced
Raman Spectroscopy (SERS) increases the Raman signal significantly
(C. R. Yonzon, C. L. Haynes, X. Zhang et al., Anal Chem 76 (1), 78
(2004); A. E. Grow, L. L. Wood, J. L. Claycomb et al., J Microbiol
Methods 53 (2), 221 (2003); K. Nithipatikom, M. J. McCoy, S. R.
Hawi et al., Anal Biochem 322 (2), 198 (2003); J. B. Jackson and N.
J. Halas, P Natl Acad Sci USA 101 (52), 17930 (2004)) through
enhanced electromagnetic fields in close proximity to a surface.
SERS measurements performed on rough metal surface or dispersed
metal nanoparticle aggregates have shown the highest Raman
enhancement factors up to 10.sup.14 for detection down to single
molecule level (Katrin Kneipp, Harald Kneipp, Irving Itzkan et al.,
Current Science (Bangalore) 77 (7), 915 (1999); S. Nie and S. R.
Emory, Science 275 (5303), 1102 (1997)) but these measurement often
suffer from poor reproducibility (M. Moskovits, Reviews of Modern
Physics 57 (3), 783 (1985)). To improve the reproducibility, other
methods including self-assembly of metallic colloidal
nano-particles (R. G. Freeman, K. C. Grabar, K. J. Allison et al.,
Science 267 (5204), 1629 (1995)), nanosphere lithography (NSL) and
metal film over nanosphere (MFON) (C. L. Haynes and R. P. Van
Duyne, Journal of Physical Chemistry B 107 (30), 7426 (2003)),
electrochemical roughening of polished gold substrate (J. M.
Sylvia, J. A. Janni, J. D. Klein et al., Analytical Chemistry 72
(23), 5834 (2000)), and periodic structured metallic substrate
using electron-beam lithography (M. Kahl, E. Voges, S. Kostrewa et
al., Sensors and Actuators B-Chemical 51 (1-3), 285 (1998)), have
been developed to fabricate SERS substrate consisting homogeneous
features over large area with reproducible enhancement factors up
to 10.sup.8. Although these efforts lead to successful utilization
of SERS analysis in many promising applications including gene and
protein discrimination (Y. C. Cao, R. C. Jin, J. M. Nam et al., J
Am Chem Soc 125 (48), 14676 (2003); Y. W. C. Cao, R. C. Jin, and C.
A. Mirkin, Science 297 (5586), 1536 (2002); T. Vo-Dinh, F. Yan, and
M. B. Wabuyele, Journal of Raman Spectroscopy 36 (6-7), 640
(2005)), bio-warfare agents detection (D. A. Stuart, K. B. Biggs,
and R. P. Van Duyne, Analyst 131 (4), 568 (2006)) and real-time
glucose monitoring (0. Lyandres, N. C. Shah, C. R. Yonzon et al.,
Analytical Chemistry 77 (19), 6134 (2005)), lacking of the ability
to fabricate SERS hot-spots at specific location limits application
for very small sample volume.
[0008] To overcome such limit, we recently developed tunable
nanoplasmonic resonators (NPRs), consisting of thin SiO.sub.2 layer
sandwiched between metallic nano-disks described in Durant S. Su K,
Steel M. J., Xiong Y. Sun C., Zhang X, Journal of Physical
Chemistry B 110 (9), 3964 (2006) hereby incorporated by reference.
The resonance frequency can be precisely tuned by varying the
dielectric layer thickness and aspect-ratio of the NPRs. Individual
NPRs can enhance the Raman intensity by a factor of
6.1.times.10.sup.10; among the largest values obtained for a single
SERS substrate or nanoparticle. Fabricated using well established
nanolithography processes, the NPR-based method enables producing
SERS hot-spots at desired location in a much smaller dimension
reproducibly, allowing multiplexed high throughput detection and
lab-on-chip applications.
[0009] Prostate cancer biomarker Prostate Specific Antigen (PSA), a
kallikrein (hK) family serine protease (S. R. Denmeade and J. T.
Isaacs, BJU Int 93 Suppl 1, 10 (2004); J. A. Clements, N. M.
Willemsen, S. A. Myers et al., Crit Rev Clin Lab Sci 41 (3), 265
(2004)), is used as a model protease in the present application.
The commonly used prostate-specific antigen (PSA) blood test has
being widely used for early diagnosis and management of prostate
cancer, the leading male cancer (H. Gronberg, Lancet 361 (9360),
859 (2003); S. R. Denmeade and J. T. Isaacs, Nat Rev Cancer 2 (5),
389 (2002)). However, serum PSA concentrations reflect the presence
of benign prostatic hyperplasia (BPH) more often than cancer (A.
Caplan and A. Kratz, Am J Clin Pathol 117 Suppl, S104 (2002); E. I.
Canto, S. F. Shariat, and K. M. Slawin, Curr Urol Rep 5 (3), 203
(2004)). The lack of specificity causes a high false-positive rate
and often leads to costly prostate needle biopsies for diagnosis
and post-biopsy complications as well as considerable anxiety (M.
B. Gretzer and A. W. Partin, Urol Clin North Am 30 (4), 677 (2003);
A. Haese, M. Graefen, H. Huland et al., Curr Urol Rep 5 (3), 231
(2004)). Recent research has identified a family of highly specific
peptides that can be cleaved by paPSA isoform in xenografts models
(S. R. Denmeade, C. M. Jakobsen, S. Janssen et al., J Natl Cancer
Inst 95 (13), 990 (2003)) and human samples (P. Wu, U. H. Stenman,
M. Pakkala et al., Prostate 58 (4), 345 (2004); P. Wu, L. Zhu, U.
H. Stenman et al., Clin Chem 50 (1), 125 (2004)) thus, measurement
of paPSA protease activity from in vivo samples is possible and
would be potentially valuable as a more specific screening agent
for prostate cancer and in detection of recurrent disease. However,
reported results based on immunopeptidemetric assays (IMPA) exhibit
low fluorescence signal-to-noise ratios, preventing reliable
measurements at lower concentrations in the clinically important
range of 60-300 pM (P. Wu, U. H. Stenman, M. Pakkala et al.,
Prostate 58 (4), 345 (2004); P. Wu, L. Zhu, U. H. Stenman et al.,
Clin Chem 50 (1), 125 (2004)). In addition, there is usually a
limited number of prostate cancer cells (<1000) isolated from
fine needle biopsy or circulating cell capture. No commercial
method exists that can perform a paPSA protease activity assay on a
small number of cells for clinical staging. Therefore, one goal of
the present invention is to provide a method that allows specific
and sensitive measurements of paPSA for prostate cancer detection
in a very small sample volume.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides the detection and measurement
of enzymatic activity in vitro and, in situ, using a nanosensor
comprised of a nanoplasmonic resonator (NPR) with at least
picomolar sensitivity. In one embodiment, a bioconjugated NPR
enhances Raman spectra intensity in Surface-Enhanced Raman
Spectroscopy (SERS) and enables sensitive single-step detection of
enzymatic activity in extremely small volume.
[0011] In certain preferred embodiments, the invention provides a
nanosensor, comprising a nanoplasmonic resonance SERS platform. The
platform comprising a substrate featuring a surface enhanced Raman
scattering (SERS) nanoplasmonic resonator singly or in an array,
wherein the nanoplasmonic resonator (NPR) has a biomolecule
conjugated thereto. In one embodiment, the NPR comprising metallic
nanodisks with alternating shielding layer(s), having a tagged
biomolecule conjugated or tethered to the surface of the NPR. In a
preferred embodiment, the tag is a Raman active tag.
[0012] In one embodiment, the biomolecule is a peptide linked to a
Raman active tag, wherein the peptide comprising a specific
sequence that can be specifically modified or cleaved by an enzyme.
Thus, this peptide-conjugated nanoplasmonic resonator is intended
to be used as a specific screening tool to provide information on
the presence, concentration and activity of enzymes such as
proteases, kinases and peptidases. In one embodiment, the screen
would measure activity of cancer biomarkers such as
prostate-specific antigen (PSA) in a biological sample. In one
embodiment, the peptides should be substrates specifically
recognized, modified and/or acted on by the corresponding enzyme to
be detected.
[0013] In another embodiment, real-time reaction monitoring also
provides critical information on enzyme activity rather than just
measuring the presence of the protein. Different Raman tag
molecules may be used such that detection of two or more types of
enzymes may be carried out by multiplexing peptide-conjugated NPRs.
Furthermore, an array of the peptide-conjugated NPRs on a substrate
is described for use to further amplify or expand detection.
[0014] In another embodiment, different biomolecule substrates
orthogonal to each other, or with minor overlap in specificity, can
be used to detect the corresponding enzyme. In one embodiment, a
biomolecule library can be conjugated to the NPRs and spatially
separated in either a random array or ordered microarray format.
The multiplexed array of the biomolecule-nanoplasmonic resonators
can be used to detect multiple enzymes simultaneously.
[0015] The NPR can also be manipulated by laser or magnetic fields
to address at high accuracy spatially, so that it could be
multiplexed as high density arrays (with sub-microliter volume).
Additional spatial multiplexing for multiple proteases in a
microarray or nanoarray format is contemplated. In addition, the
magnetic or laser maneuverability allow biosensing at desired
locations, which would be useful for obtaining in situ measurements
intracellularly.
[0016] In another embodiment, the nanosensor herein described may
be integrated into microfluidics system or other chip system. In
one embodiment, the nanosensor-based assay can be performed in
liquid phase, wherein the sample is contacted with the NPR
nanosensor or the NPR array and the SERS measurements can be
conducted.
DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 Schematic illustration of the working principle of
detecting PSA protease activity using peptide-conjugated NPR SERS
nanosensors. (a) NPRs exhibit a tunable plasmon resonance and
highly enhanced local electromagnetic field through coupled
plasmonic resonance. NPRs with a short axis of 150 nm and long axis
of 200 nm were made of multi-stacks of silver and SiO.sub.2 layers
with thicknesses of 25 nm and 5 nm, respectively. (b) The molecular
structure of the biomarker that consists of Raman dye R19, PSA
specific peptide sequence HSSKLQLAAAC (SEQ ID NO:1), and cysteine.
The peptide can be cleaved by PSA enzyme between HSSKLQ and LAAAC.
(c) The detection scheme of NPR functionalized with peptide
sequence HSSKLQLAAAC (SEQ ID NO:1) and the Raman dye R19 (star).
The presence of the PSA enzyme will cleave the peptide sequence.
After cleavage, the diffusion of the R19 (star) away from the
surface will be monitored by the loss of the SERS signatures of the
R19 moiety. Packing molecule octanethiol
(HS--(CH.sub.2).sub.2--CH.sub.3) was used to reduce the packing
density of the reporting peptide on NPR surface, and thus, allows
PSA enzyme to access the reporting peptide. (d) Optical microscopic
image of NPRs arrays fabricated using standard e-beam lithography
and thin film deposition process. Fabricated NPRS arrays consists
of 30.times.30 NPRs with 500 nm spacing. Multiple NPRs arrays and
alignment mark can be conveniently fabricated on the same
substrate. (e) Magnified image of NPR array measured by Scanning
Electron Microscope. Using precision lithography methods, the NPR
can be prepared in a controlled manner. (f) Image of NPRs measured
at higher magnification using Atomic Force Microscope (AFM). (g)
Measured extinction spectrum of an NPR array at a wavelength range
of 425 to 650 nm. The resonance peak of the NPR has been tuned to
closely match the laser excitation and Raman emission frequencies,
and thus, maximize the overall enhancement of the Raman signal. (h)
Highly reproducible Raman spectra of para-mercaptoaniline (pMA)
molecules conjugated on NPR surface measured at different NPRs
arrays. Integration time is 30 seconds.
[0018] FIG. 2 Real-time kinetic measurement of PSA protease
activity. (a) SERS spectra for 6 nM PSA incubation taken over 30
minutes with an integration time of 30 seconds. (b) SERS spectra
for the negative control, 6 nM granzyme B, taken over 30 minutes.
(c) Time-resolved measurements of relative change in Raman peak
intensity at 1316 cm.sup.-1, 1456 cm.sup.-1, 1526 cm.sup.-1, and
1597 cm.sup.-1 before and after addition of PSA protease. Negative
time represents time before protease addition.
[0019] FIG. 3 Time-resolved measurements of PSA activity by varying
the active PSA concentration. (a) Normalized SERS intensity change
for 1316 cm.sup.-1 peak at active PSA concentration from 6 pM to 6
nM. The decreasing of SERS intensity can be clearly measured while
no significant change can be observed in the control experiment
with no active PSA protease. (b) Concentration dependence of
normalized SERS intensity change obtained at 30 min. (c) Protease
activity measurement obtained from unprocessed extracellular fluid
(ECF): Raman spectra obtained at the beginning of exposing LNCaP
cells (positive control) ECF to NPR nanosensors (t=0 min) and after
30 minutes. (d) Normalized change of SERS intensity at 1316
cm.sup.-1 peak indicating PSA proteolytic activity in the fluid
extracted from LNCaP and K562 cell lines.
[0020] FIG. 4. PSA detection scheme using Raman detection and
spectroscopy.
[0021] FIG. 5A. A SERS microspectroscopy system and nanoplasmonic
resonator visualization and real-time enzyme reaction detection.
FIG. 5B. A schematic of an optical spectrum measurement setup.
[0022] FIG. 6 (A) Simulated scattering spectra for single nanodisks
with various metal layers. The nanodisk is circular with a 90 nm
diameter, while the total thicknesses for metal layers and
SiO.sub.2 layers are kept both at 30 nm. The polarization of the
incident light is polarized in the Y-axis direction. (B) Y-Z plane
cross section of local electrical field distribution for the single
Au layer nanodisk (SiO.sub.2/Au/SiO.sub.2) at the resonant
frequency. (C) Y-Z plane cross section of local electrical field
distribution for the 6 Au layer nanodisks (SiO2/Au).sup.6/SiO.sub.2
at resonant frequency.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0023] The present invention demonstrates the in vitro detection
and measurement of enzymatic activity using a nanosensor comprised
of a nanoplasmonic resonator (NPR) with at least picomolar
sensitivity. In one embodiment, a bioconjugated NPR enhances Raman
spectra intensity in Surface-Enhanced Raman Spectroscopy (SERS) and
enables sensitive single-step detection of enzymatic activity in
extremely small volume.
[0024] One of the major advantages and applications of the small
volume property is that it is useful in detecting proteases such as
prostate-specific antigen (PSA) activity of cancer cells at single
cell level. The small volume requirement and sensitivity level
makes it possible to detect PSA activity in captured circulating
prostate cancer cells for indications of various disease states,
e.g., metastasis, which is not feasible with conventional
techniques. In semen, the PSA concentration is 10-150 .mu.M, with
approximately two thirds of the PSA enzymatically active. The
sensitivity level achieved with the NPR PSA probe (nanomolar range)
is sufficient for a seminal fluid based assay, thus the
nanoplasmonic resonance SERS platform described herein is intended
to have clinical applications.
[0025] In a preferred embodiment, the invention provides a
nanosensor, comprising a nanoplasmonic resonance SERS platform. The
platform comprising a substrate featuring a surface enhanced Raman
scattering (SERS) nanoplasmonic resonator singly or in an array,
wherein the nanoplasmonic resonator (NPR) has a biomolecule
conjugated thereto. In one embodiment, the NPR comprising at least
two nanodisks with an alternating thin shielding layer(s), and a
tagged biomolecule conjugated or tethered to the surface of the
NPR. In a preferred embodiment, the tag is a Raman active tag.
[0026] A variety of detection units of potential use in Raman
spectroscopy are known in the art and any known Raman detection
unit may be used. A non-limiting example of a Raman detection unit
is disclosed in U.S. Pat. No. 6,002,471. In this example, the
excitation beam is generated by either a Nd:YAG laser at 532 nm
(nanometer) wavelength or a Ti:sapphire laser at 365 nm wavelength.
Pulsed laser beams or continuous laser beams may be used. The
excitation beam passes through confocal optics and a microscope
objective, and may be focused onto a substrate containing attached
biomolecule targets. Raman emission light target(s) can be
collected by the microscope objective and the confocal optics,
coupled to a monochromator for spectral dissociation. The confocal
optics can include a combination of dichroic filters, barrier
filters, confocal pinholes, lenses, and mirrors for reducing the
background signal. Standard full field optics can be used as well
as confocal optics.
[0027] The Raman emission signal can be detected by a Raman
detector. The detector can include an avalanche photodiode
interfaced with a computer for counting and digitization of the
signal. Where arrays of target(s) are to be analyzed, the optical
detection system may be designed to detect and localize Raman
signals to specific locations on a chip or grid. For example,
emitted light may be channeled to a CCD (charge coupled device)
camera or other detector that is capable of simultaneously
measuring light emission from 20 multiple pixels or groups of
pixels within a detection field.
[0028] Various excitation sources include, but are not limited to,
a nitrogen laser (Laser Science Inc.) at 337 nm and a
helium-cadmium laser (Liconox) at 325 nm (U.S. Pat. No. 6,174,677).
The excitation beam can be spectrally purified with a bandpass
filter 30 (Corion) and may be focused on a substrate 140 using a
6.times. objective lens (Newport, Model L6X). The objective lens
can be used to both excite the indicator(s) and to collect the
Raman signal, by using a holographic beam splitter (Kaiser Optical
Systems, Inc., Model HB 647-26NI8) to produce a right-angle
geometry for the excitation beam and the emitted Raman signal. A
holographic notch filter (Kaiser Optical Systems, Inc.) can be used
to reduce Rayleigh scattered radiation. Alternative Raman detectors
include, but are 5 not limited to, an ISA HR-320 spectrograph
equipped with a red-enhanced intensified charge-coupled device
(RE-ICCD) detection system (Princeton Instruments). Other types of
detectors may be used, such as charged injection devices,
photodiode arrays or phototransistor arrays
[0029] Referring now to FIG. 1A, the nanoscale dimension and the
high local electromagnetic field enhancement of the NPR enables a
high-sensitivity optical detection of biomolecular reactions on its
surface. In a preferred embodiment, the nanoplasmonic resonators
are lithographically defined metallodielectric nanoparticles
comprising at least two nanodisks stacked vertically, separated by
a shielding layer. In various embodiments, the NPR are preferably
patterned on a substrate by electron beam lithography or other
lithographic methods known in the art.
[0030] The substrate on which the NPRs are patterned can be
comprised of quartz, polystyrene, silica, dextran, or any other
materials with constant Raman spectra. In one embodiment, to
prevent charging effects during the electron beam lithography, the
substrate is further coated with a layer such as indium tin oxide.
The layer can be sputtered, deposited coated or added using any
other method onto the substrate to form a thin film. In another
embodiment, the substrate is then spin-coated with a polymer to
create a positive photoresist before exposure to create the
patterns. In one embodiment, after exposure, patterns are developed
using a solvent mixture, followed by multilayer electron beam
evaporation and standard lift-off procedure.
[0031] In one embodiment, the NPRs are made as described in Example
1. In another embodiment, the NPRs are made as described in K. H.
Su, Q. H. Wei, and X. Zhang, "Tunable and augmented plasmon
resonances of Au/SiO.sub.2/Au nanodisks", Appl. Phys. Lett. 88,
063118, 2006; and Kai-Hung Su, Stephane Durant, Jennifer M. Steele,
Yi Xiong, Cheng Sun, and Xiang Zhang, Raman Enhancement Factor of a
Single Tunable Nanoplasmonic Resonator, Journal of Physical
Chemistry B, 110 (9), 3964 (2006), both of which are hereby
incorporated by reference in their entirety.
[0032] In a preferred embodiment, the nanoplasmonic resonator is
comprised of nanodisks layered or stacked with an alternating
shielding layer. For example, NPRs can be made having two nanodisks
of gold with a shielding layer of SiO.sub.2 sandwiched in between.
In another embodiment, the two nanodisks of gold with a shielding
layer of SiO.sub.2 sandwiched in between is capped on one end with
another shielding layer of SiO.sub.2, thereby producing an
SiO.sub.2/Au/SiO.sub.2/Au nanoplasmonic resonator.
[0033] In one embodiment, the nanoscale layers are evaporated on
the patterned substrate to form the nanodisk layers of the NPRs. In
a preferred embodiment, each nanodisk is 50-500 nm at its widest
point. In other embodiments, each nanodisk is 10-500 nm at the
widest point. The nanodisks can be comprised of a thin layer of a
metal, a semiconductor material, multi-layers of metals, a metal
oxide, an alloy, a polymer, or carbon nanomaterials. In certain
embodiments the nanocrescent comprises a metal selected from the
group consisting of Ga, Au, Ag, Cu, AI, Ta, Ti, Ru, Ir, Pt, Pd, Os,
Mn, Hf, Zr, V, Nb, La, Y, Gd, Sr, Ba, Cs, Cr, Co, Ni, Zn, Ga, In,
Cd, Rh, Re, W, Mo, and oxides, and/or alloys, and/or mixtures,
and/or nitrides, and/or sintered matrix thereof.
[0034] The shielding layer of material can be any material having a
constant Raman spectra. The shielding layer functions as a tuning
parameter for plasmon resonant frequency of the NPR. Compared to
single layered metallic nanodisks, multilayered nanodisks exhibit
several distinctive properties including significantly enhanced
plasmon resonances and tunable resonance wavelengths which can be
tailored to desired values by simply varying the dielectric layer
thickness while the particle diameter is kept constant. Numerical
calculations show that slicing one metal layer into metal
multilayers leads to higher scattering intensity and more "hot
spots," or regions of strong field enhancement. FIG. 6 shows a
color simulated scattering spectra for nanodisks with various metal
layers.
[0035] Thus, in another embodiment, each nanodisk layer in the NPR
can be the same or different thickness. By choosing different layer
thicknesses, the plasmon resonance wavelength and the surface
enhancement factor can be tuned to match various applications. For
instance, in Example 1, NPRs with a short axis of 150 nm and long
axis of 200 nm were made of multi-stacks of silver and SiO.sub.2
layers with thicknesses of 25 nm and 5 nm, respectively.
Furthermore, by selective shielding of the outer surface of the
metallic structure, the efficiency can be further enhanced by
guiding the molecular assembly to the locations that exhibit strong
electromagnetic fields. Thus, in another embodiment, the outer
layers are shielding layers comprising material having a constant
Raman spectra. FIG. 1A shows the schematics and transmission
electron micrograph of a preferred nanoplasmonic resonator
(NPR).
[0036] In one embodiment, the biomolecule is a peptide comprising a
specific sequence that can be specifically cleaved by protease,
linked to a Raman active tag.
[0037] A variety of Raman labels are known in the art (e.g., U.S.
Pat. Nos. 5,306,403; 6,002,471; 6,174,677, which are incorporated
herein by reference) and any such known Raman label(s) can be used.
The labels typically have characteristic (e.g., unique) and highly
visible/detectable optical signatures. Suitable Raman labels
include, but are not limited to fluorophore, a chromophore, a
quantum dot, a fluorescent microsphere, biotin, and the like. In
certain embodiments the Raman label comprises a Rhodamine, a
fluoresceine, or an exogenous chemical molecule. In certain
embodiments the Raman label comprises a moiety acting as a Raman
tag. Non-limiting examples of tag molecules include TRIT
(tetramethyl rhodamine isothiol), NBC
(7-nitrobenz-2-oxa-1,3-diazole), Texas Red dye, phthalic acid,
terephthalic acid, isophthalic acid, cresyl fast violet, cresyl
blue violet, brilliant cresyl blue, para-aminobenzoic acid,
erythrosine, biotin, digoxigenin,
5-carboxy-4',5'-dichloro-2',7'-dimethoxy fluorescein,
5-carboxy-2',4',5',7'tetrachlorofluorescein, 5-carboxyfluorescein,
5-carboxy rhodamine, 6-carboxyrhodamine, 6-10 carboxytetramethyl
amino phthalocyanines, 6-carboxy-X-rhodamine, azomethines,
cyanines, xanthines, succinylfluoresceins, aminoacridine, and
cyanide (CN), thiol (SH), chlorine (el), bromine (Br), methyl,
phorphorus (P), sulfur (S), SN, AI, Cd, Eu, Te, and compounds
containing such moieties. In certain embodiments, carbon nanotubes,
quantum dots (see, e.g., Evident Technologies, Troy N.Y.;
Invitrogen/Molecular Probes, 15 etc.), or microspheres (e.g.
fluorescent microspheres (see, e.g., Transfluosphres.RTM. from
InvitrogenIMolecular Probes) can be used as Raman tags.
[0038] In various embodiments, one or more Raman labels (Raman
tags) can be attached to the biomolecule (e.g., polypeptide) that
is attached to the NPR(s). The presence of such Raman tags can
enhance the change in Raman signal produced by cleavage of the
peptide.
[0039] Thus, in one embodiment this Raman tagged
biomolecule-conjugated nanoplasmonic resonator is intended to be
used as a specific screening tool to provide information on the
presence, concentration and enzymatic activity of enzymes and other
cancer biomarkers, such as prostate-specific antigen (PSA) in a
biological sample. In certain embodiments, the biomolecule is a
peptide. The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer. In certain embodiments, multiple peptides are
conjugated to the surface of the nanoscrescent, each being the same
or different. In various embodiments approximately 5 to 500, more
preferably about 10 to about 400, still more preferably about 20,
30, or 40 to about 200, 250, or 300, and most preferably about 50
to about 150 substrate molecules (e.g. peptides) are attached to
the nanocrescent. In one embodiment, about 100 peptides are 15
conjugated to the nanocrescent with direct reaction between Au and
the thiol group on the Peptide.
[0040] The enzyme whose activity is being monitored can include but
is not limited to, an enzyme, protease, kinase, peptidase or other
biological molecule. In various embodiments, the biomolecules are
peptides specifically recognized and modified or cleaved by the
corresponding enzyme to be detected. In another embodiment, the
biomolecules are peptides specifically recognized and
phosphorylated by the kinase to be detected. Various types of
proteases and peptides specifically recognized by those proteases
are also described in co-pending International Application No.
PCT/US2007/010722, entitled "Detection of Protease and Protease
Activity Using a Single Nanoscrescent SERS Probe." Related methods
using a nanocrescent probe for SERS detection of a protease or
other biomolecules is also described in PCT/US2007/010722, which is
hereby incorporated by reference for all purposes.
[0041] In a preferred embodiment, the biomolecule is a peptide,
wherein the peptide is an oligopeptide about 10-12 amino acid
residues in length. However, the peptide can be as short as 4 amino
acid residues, and as long as 100 amino acids. In one embodiment,
the peptides should be sequences specifically recognized and
modified by a corresponding enzyme. The peptide can be synthesized
and obtained commercially or the peptides can be made according to
the methods described in Example 1. Raman active molecules such as
biotin or Rhodamine 6G (R19) (FIG. 1B) are preferably grafted
through a short polyethyleneglycol or aminovaleric acid linker at
the amino terminus of the peptide.
[0042] The biomolecules may be "conjugated" (i.e., linked) to the
nanoplasmonic resonator directly or via one or more linking agents.
"Linking agent" as used herein refers to any compound that forms a
bond between the NPR and the biomolecule and include e.g., a
functional group, an affinity agent, or a stabilizing group.
Suitable bonds include ionic interactions, covalent chemical bonds,
physical forces such van der Waals or hydrophobic interactions,
encapsulation, embedding, binding affinity, attraction or
recognition, and various types of primary, secondary, tertiary
linkages including but not limited to, peptide, ether, ester,
acryl, aldehyde, ketone, acryloyl, thiol, carboxyl, hydroxyl,
sulfhydryl and amine linkages or the like.
[0043] In one embodiment, hundreds of peptides are conjugated to
the NPR with direct reaction between the metallic nanodisk of the
NPR and a thiol group on the peptide. In another embodiment, the
NPR metallic surface can also be modified with either amine or
carboxyl group so the peptide can be tethered through peptide bond
on the NPR surface, via reaction with a heterobifunctional
crosslinker that can react with both amine group and thiol group,
or carboxyl group and thiol group. In a specific embodiment, an
Au/SiO.sub.2/Au/SiO.sub.2 NPR can be functionalized with amine or
carboxyl functional groups, and the peptides can be crosslinked via
the crosslinker to the amine and carboxyl functional groups.
Various crosslinkers can be used for the conjugation of
thiol-activated peptides, and the surface functional groups on the
NPRs, such as amine, carboxyl, and hydroxyl groups.
[0044] In one preferred embodiment, the substrate peptide is
tethered onto the surface of an Au/SiO.sub.2/Au NPR using a
cysteine group at the carboxyl terminus of the peptide to attach
the peptide to the Au surface, relying on the Au-thiol reaction to
form a covalent bond. In a preferred embodiment, multiple peptides
are similarly conjugated to the surface of the NPR, each being the
same or different.
[0045] The NPR indicators described herein can utilize polypeptide
sequences comprising one or more recognition site(s) for any
protease(s) it is desired to detect. Proteases (proteolytic
activity) are not only required for maintenance of normal cellular
functions but are also central to the pathogenesis of a variety of
human diseases. Parasitic (for example schistosomiasis and
malaria), fungal (such as C. albicans) and viral infections (for
example HIV, herpes and hepatitis), and also cancer, inflammatory,
respiratory, cardiovascular and neurodegenerative diseases,
including Alzheimer's, require proteolytic activity for progress.
Detection of protease presence, quantity, or activity is thus
useful as a diagnostic/prognostic marker for the presence or
likelihood of disease. In addition, detection of protease activity
(or the inhibition thereof) is useful in screening for protease
inhibitor therapeutics for the treatment of a number of
pathologies.
[0046] A "protease" that can be detected and/or quantified
according to the invention is an enzyme that typically hydrolyzes a
peptide bond between a pair of amino 20 acids located in a
polypeptide chain, also called an endoprotease. Proteases are
typically defined by reference to the nucleophile in the catalytic
center of the enzyme. The most common nucleophiles arise from the
side chains of serine, aspartic acid, and cysteine, resulting in
families of proteases, such as serine proteases (Paetzel et al.
(1997) Trends Biochem. Sci. 22: 28-31), aspartyl proteases
(Spinelli et al. (1991) Biochemie 73: 1391-25 1396), and cysteine
proteases (Altschuh et al. (1994) Prot. Eng. 7: 769-75,1994).
Metalloproteases usually contain a zinc catalytic metal ion at the
catalytic site (Klimpel et al. (1994) Mol. Microbiol. 13:
1093-1100).
[0047] A "protease recognition site" is a contiguous sequence of
amino acids connected by peptide bonds that contains a pair of
amino acids which is connected by a peptide bond that is hydrolyzed
by a particular protease. Optionally, a protease recognition site
can include one or more amino acids on either side of the peptide
bond to be hydrolyzed, to which the catalytic site of the protease
also binds (Schecter and Berger, (1967) Biochem. Biophys. Res.
Commun. 27: 157-62), or the recognition site and cleavage site on
the protease substrate can be two different sites that are
separated by one or more (e.g., two to four) amino acids.
[0048] The specific sequence of amino acids in the protease
recognition site typically depends on the catalytic mechanism of
the protease, which is defined by the nature of the functional
group at the protease's active site. For example, trypsin
hydrolyzes peptide bonds whose carbonyl function is donated by
either a lysine or an arginine residue, regardless of the length or
amino acid sequence of the polypeptide chain. Factor Xa, however,
recognizes the specific sequence Ile-Glu-Gly-Arg and hydrolyzes
peptide bonds on the C-terminal side of the Arg. Various preferred
protease recognition sites include, but are not limited to protease
recognition sites for proteases from the serine protease family, or
for metalloproteases, or for a protease from the cysteine protease
family, and/or the aspartic acid protease family, and/or the
glutamic acid protease family. In certain embodiments preferred
serine proteases recognition sites include, but are not limited to
recognition sites for chymotrypsin-like proteases, and/or
subtilisin-like proteases, and/or alpha/beta hydrolases, and/or
signal peptidases. In certain embodiments preferred metalloprotease
recognition sites include, but are not limited to recognition sites
for metallocarboxypeptidases or metalloendopeptidases. Illustrative
proteases and protease recognition sites are shown below in Table
1.
TABLE-US-00001 TABLE 1 Illustrative proteases and protease
recognition sites (*indicates the peptide bond being hydrolyzed).
Protease Family Protease Protease Recognition Sites serine factor
Xa Ile-Gly-Gly-Arg* serine trypsin Lys*, Arg* serine chymotrypsin
Tyr*, Phe*, Leu*, Ile*, Val*, Trp*, and His* at high pH serine
thrombin Arg* serine PSA serine and peanut mottle polyvirus
Glul-Xaa-Xaa-Tyr- cysteine Nla protease Gln*(Ser/Gly) variants
cysteine papaine Arg*, Lys*, Phe* cysteine bromelaine Lys*, Ala*,
Tyr*, Gly* cysteine cathepsin B Arg*Arg, Phe*Arg cysteine cathepsin
L Phe*Arg aspartyl HIV protease Phe*Pro aspartyl S. cerevisiae
yapsin 2 Lys*, Arg* aspartyl cathepsin D Phe*Phe Phe*Lys Leu*Phe
Leu*Tyr metallo- thermolysin *Tyr, *Phe, *Leu, *lle, *Val, Trp, and
*His metallo- peptidyl-Lys Xaa*Lys metalloendopeptidase metallo-
peptidyl-Asp Xaa*Asp metallodndopeptidase Xaa*Glu Xaa*Cys metallo-
coccolysin *Leu, *Phe, *Tyr, *Ala metallo- autolysin
Leu-Trp-Met*Arg-Phe-Ala metallo- gelatinase A (MMP-2)
Pro-Gln-Gly*Ile- Ala-Gly-Gln metallo- human neutrophil
Gly-Leu-Ser-Ser- collagenase (MMP-8) Asn-Pro*Ile-Gln-Pro
[0049] In a specific embodiment to detect PSA, the peptide design
will follow the amino acid sequence of the active site of
PSA-specific peptides with serine residues and flanking sequences
that can be recognized by PSA. In a preferred embodiment, the
peptide contains the sequence of HSSKLQ-LAAAC (SEQ ID NO:1) which
has been shown to have very high specificity for proteolytically
active PSA. (See Denmeade, S. R. et al. Specific and efficient
peptide substrates for assaying the proteolytic activity of
prostate-specific antigen. Cancer Res 57, 4924-4930 (1997)). It has
been shown that HSSKLQ-L is cleaved by PSA but not by any other
proteases in vivo in a mouse model. Thus, in another embodiment,
multiple peptides can be generated, each having a random or known
sequence portion, so long as each incorporates the highly specific
sequence of HSSKLQ-LAAAC (SEQ ID NO:1).
[0050] The PSA digestion site is between the Glutamine (Q) and
Leucine (L) residues in the peptide HSSKLQ-LAAAC (SEQ ID NO:1), and
the peptides are digested into 2 fragments, HSSKLQ and LAAAC. In
the present preferred embodiment, the peptide is preferably
tethered to the NPR surface as shown in FIG. 1B, such that the PSA
peptide is not sterically hindered from the PSA enzyme and thereby
optimally accessible, and the Raman tag, R19, is attached to the
peptide at the Histidine. It is contemplated that an additional
spacer synthesized in between the peptide sequence HSSKLQ-LAAAC
(SEQ ID NO:1) and the NPR after the Cys (C) residue, will improve
the presentation of PSA substrate peptide HSSKLQ on the surface and
thereby increase the detection sensitivity. Although by doing so
the distance of the Raman tag molecules could be farther from the
NPR surface and resulting in lower Raman intensity level because
the coil-like short peptide structure implies large chance for the
distal Raman tag molecule to contact the metal surface of the NPR.
In a preferred embodiment, the spacer is a packing molecule such as
octanethiol (HS--(CH.sub.2).sub.2--CH.sub.3) and used to reduce the
packing density of the reporting peptide on NPR surface, and thus,
allows the enzyme access to the reporting peptide.
[0051] The present approach can be used to detect the presence of
other hydrolytic biological molecules. Thus, for example the
peptide protease substrate can be replaced with single or
double-stranded nucleic acids (RNA or DNA), and the indicator can
detect and/or quantify the presence of active nucleases. In such
instances, the nucleic acid substrate will typically comprise one
or 30 more recognition sites for nucleases (e.g. restriction
endonucleases). The nuclease recognition sites typically range in
length from about 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp or 10 bp
to about 15 bp, 20 bp, 25 bp, or 30 bp. In various embodiments the
nucleic acid can range in length from about 3 bp to about 200 bp,
preferably from about 4 bp to about 100 bp, more preferably from
about 6, 8, 10, 16, or 20 bp to about 80, 60, 40, or 30 bp.
[0052] In another embodiment, essentially any molecule that can be
phosphorylated by a kinase can be used as a kinase substrate in the
methods and compositions described herein. While proteins/peptides
comprise the largest substrate class for kinases, a number of other
kinase substrates are known as well. Such substrates include, but
are not limited to various sugars (e.g., hexose, glucose, fructose,
mannose, etc.), nucleotides/nucleic acids, acetate, butyrate, fatty
acids, sphinganine, diacylglycerol, ceramide, and the like.
Illustrative protein kinase substrates and the sequences is
described in the sequence listing and Table 3.
TABLE-US-00002 TABLE 3 Illustrative protein kinase substrates. SEQ
ID Kinase Substrate NO cAMP-dependent protein kinase LRRASLG
(Kemptide) 2 cAMP dependent protein kinase GRTGRRNSI 3 (PKA)
protein kinase C (PKC) QKRPSQRSKYL 4 protein kinase Akt/PKB RPRAATF
5 Abl kinase EAIYAAPFAKKK 6 5'-AMP-activated protein kinase
HMRSAMSGLHLVKRR 7 (AMPK) Ca2+/calmodulin-dependent KKALRRQETVDAL
(Autocamtide-2) 8 protein kinase cyclin-dependent kinase 2
(Ac-S)PGRRRRK 9 (cdc2) cyclin-dependent kinase 2 HHASPRK 10 (Cdk2)
cyclin-dependent kinase 5 PKTPKKAKKL 11 (Cdk5) casein kinase 1
(CK1) RRKDLHDDEEDEAMSITA 12 CK2 alpha subunit or RRRDDDSDDD 13
holoenzyme DYRK family protein kinases KKISGRLSPIMTEQ 14 GSK3 alpha
and beta YRRAAVPPSPSLSRHSSPHQ(pS)EDEEE 15 Src kinase
KVEKIGEGTYGVVYK 16 checkpoint kinases CHK1 and
KKKVSRSGLYRSPSMPENLNRPR 17 CHK2 protein tyrosine kinases (PTKs)
Poly(Glu:Tyr).sub.4:1 is sodium salt polymer in phosphorylation
assays. with a random amino acid distribution and a molar ratio of
4:1 for glutamic acid:tyrosine
[0053] Thus, for example, a protein, and/or sugar, and/or complex
carbohydrate, and/or lipid, and/or nucleic acid "substrate" can be
provided coupled to one or more NPRs when the substrate is
recognized and bound by a cognate binding partner, the Raman
spectrum will be changed and the interaction is detected.
[0054] A typical experimental system configuration is shown in FIG.
6, comprising a microscopy system with Raman spectrometer used to
acquire Raman scattering spectra from single tagged nanoplasmonic
resonators. In a preferred embodiment, the system is comprised of
inverted microscope equipped with a digital camera and a
monochromator with a spectrograph CCD camera, a laser source and an
optical lens. In one embodiment, Raman spectra can be measured
using a modified inverted microscope, such as the Carl Zeiss
Axiovert 200 (Carl Zeiss, Germany), with a 50.times. objective in a
backscattering configuration. The laser wavelength can be in the
visible and near infrared region. In a preferred embodiment, a 785
nm semiconductor laser is used as the excitation source of Raman
scattering, and the laser beam is focused by a 40.times. objective
lens on the NPR. The 785 nm or other near infrared light source can
assure less absorption by the biological tissue and lower
fluorescence background. However, for certain applications, lower
wavelength excitation light might be more advantageous, and even UV
light excitation can be used for applications. The excitation power
can also be measured by a photometer to insure an output of
.about.0.5 to 1.0 mW. The Raman scattering light is then collected
through the same optical pathway through a long-pass filter and
analyzed by the spectrometer. The Raman spectrometer is preferably
linked to a computer whereby the spectrometer can be controlled and
the spectra can be obtained and a spectrograph can be observed. The
spectral detection can be done with ordinary spectral polychrometer
and cooled CCD camera. The monitored wavenumbers of Raman peaks
range from 400 cm.sup.-1 to 2000 cm.sup.-1.
[0055] In one embodiment, the peptide-conjugated NPRs are incubated
with a sample suspected of containing the biomolecule to be
detected, preferably in a closed transparent microchamber. The
microchamber is mounted on a 37.degree. C. thermal plate on an
inverted Raman microscope with darkfield illumination for
nanoparticle visualization. The NPRs are visualized using the
darkfield illumination from oblique angles as the bright dots shown
in the inset pictures in FIG. 1D. The excitation laser is focused
on the NPRs by a microscopy objective lens. The SERS signal is
collected by the same objective lens and analyzed by a
spectrometer. The pictures in FIG. 1D show the .about.0.8 mW
excitation laser spot focusing on a field of NPRs.
[0056] The real-time detection of enzymatic reactions can occur
within 30 minutes. However, the incubation and detection can be as
short as 1 to 5 minutes and as long as 24 hours, or longer, if the
application needs longer incubation time biologically. After
initial centrifugal fractionation, the soluble content in
biological sample can be directly incubated with the NPRs or the
NPR array. In one embodiment, to specifically inhibit the
protease-mediated proteolysis of the conjugated peptides, protease
inhibitors are introduced prior to the addition of the protease.
For example, the peptide digestion by PSA is more than 90%
suppressed after the addition of inhibitors given the same
experimental conditions.
[0057] In various embodiments the enzyme presence, and/or
concentration, and/or activity is determined in a biological
sample. The biological sample can include essentially any
biomaterial that it is desired to assay. Such biomaterials include,
but are not limited to biofluids such as blood or blood fractions,
lymph, cerebrospinal fluid, seminal fluid, urine, oral fluid and
the like, tissue samples, cell samples, tissue or organ biopsies or
aspirates, histological specimens, and the like.
[0058] One typical experimental detection scheme for enzyme
presence, concentration and activity is shown in FIG. 5. In the
method, a solution or sample is provided to a peptide-conjugated
SERS NPR or NPR array. Before the enzymatic reaction, the SERS
spectrum of a peptide-conjugated NPR contains the characteristic
peaks from the Raman tag molecules, the peptides, and the
nanoplasmonic resonator. The enzymatic reaction by the enzyme
should modify the peptide at a predetermined modification site. For
example, during the digestion reaction by PSA, the peptide HSSKLQ-L
is cleaved between the Q and L residues, here denoted by a dashed
line. The SERS spectra of the artificial peptides change after
cleavage by the protease because the cleavage fragment containing
the Raman tag molecules diffuses away from the NPR surface, while
the other fragment remains on the NPR surface. The characteristic
SERS peaks of the molecular moieties with the Raman active tag
disappear due to the diffusive dislocation of the tag molecules
from the NPR surface into the solution after peptide digestion;
therefore the existence and concentration of the proteolytically
active PSA in solution can be probed by monitoring the SERS spectra
of the peptide-conjugated NPRs. The Raman scattering signal of the
attached peptide is then amplified by the NPR and detected by a
microscopy system as described comprising a Raman spectrometer to
acquire Raman scattering spectra from single or arrayed NPRs. For
example, as shown in FIG. 1g, the measured resonance peak of
NPRs-peptide-R19 conjugates closely matches laser excitation
wavelength at 532 nm and thus, maximizes the enhancement of Raman
scattering. As shown in FIG. 1h, NPR-based SERS substrate exhibits
reproducible Raman spectrum with consistent enhancement factor at
same order of magnitude. The variation of experimentally measured
the SERS intensities obtained from 6 different NPR array are below
25% and it can be easily normalized in the experiment.
[0059] The reaction dynamics can be monitored by time-resolved SERS
spectra acquisitions. In one embodiment, the assay can be performed
by exposing the biomolecule-conjugated NPR array to biological
fluidic samples and the subsequent time-dependent R19 Raman spectra
change is recorded at an interval of about one minute and an
integration time of about 30 seconds. The Raman peak at 1316
cm.sup.-1 of SERS label molecule (R19) can be monitored as the
primary signature peak, while the other signature peaks such as
1456 cm.sup.-1, 1526 cm.sup.-1, and 1597 cm.sup.-1 peaks, may be
also monitored as additional references (see FIG. 2). The
time-resolved spectral measurement in the presence of an enzyme can
be plotted as in FIG. 2c. The SERS intensities are proportional to
any remaining biomolecules on the NPR surface that have not been
acted on by the enzyme, thus making the normalized SERS intensity
change a direct indicator of enzyme activity. Despite the variation
in the initial intensity of different Raman signature peaks, the
normalized data all converge into the same curve, indicating the
normalized SERS intensity change is a reliable measure to
quantitatively determine the peptide being profiled or
detected.
[0060] For example, as shown in FIG. 2c, before enzyme addition,
the peptide-conjugated NPR shows steady intensities for all the
peaks over a 10 minute period. However, upon addition of an enzyme,
a significant decrease in each Raman peak is observed in the first
10-12 minutes, indicating that the enzyme is active and was able to
cleave the peptides on the NPR. At the endpoint of 30 minutes, the
decrease of the Raman signal reaches a plateau at PSA concentration
of 6 nM while at lower concentration level (.about.pM), the signal
continues to decrease at a much lower rate. Another serine
protease, Granzyme B, was selected as a negative control (FIG.
2b-c). Within 50 minutes of recording, no substantial changes in
SERS intensity was observed, even at concentrations up to 1 .mu.M.
This result demonstrates that the decrease of Raman signal in the
PSA assay was based on a genuine enzymatic process, rather than a
non-specific hydrolysis reaction or due to the displacement of the
peptide from the surface by other components in the buffer
[0061] In a preferred embodiment, the time-lapse intensities of the
Raman peak of the Raman active tag in the NPR SERS probe in the
digestion reaction is obtained with the protease, the protease with
inhibitor, and a negative control, respectively. All the peak
intensity values are normalized to the internal control peak (e.g.,
In FIG. 1g, the peak intensity measured for the R19-peptide and NPR
is 532 cm.sup.-1) and the initial peak intensity at the wavenumber
of either the positive or negative control. In one embodiment, the
negative control is a NPR-peptide hybrid, in which the peptide is
not a substrate of the protease(s) of interest and would not be
cleaved by the protease(s) being studied. The results should
indicate that the peptides are efficiently and specifically cleaved
by PSA by the gradual disappearance of the peak intensity of the
Raman active tag.
[0062] The NPR particle serves as the Raman signal amplifier and
the detected Raman signal comes from all the peptides tethered on
the surface of the NPR particle. In one embodiment, at least 100
peptide molecules are attached per NPR. Even if this number of
peptides is attached, it is contemplated that the NPR surface with
the highest SERS signal is not fully taken advantage of, if a small
percentage of the peptides are attached to the region that provides
the greatest enhancement in electromagnetic field (FIG. 1C). The
numerical simulation (FIG. 1c) indicates the amplitude of the local
electric field can be enhanced by close to 20 dB (100 fold)
especially around the edge of the nanodisk. Due to the fourth power
relation between the electric field amplitude and the Raman
enhancement factor, the peptide Raman signal could be amplified
several fold (e.g. 10.sup.8) by the NPR.
[0063] Furthermore, because tens to hundreds of peptides are used
in the conjugation reaction for each NPR on average, the
disappearance of the characteristic Raman peaks from the tag
molecules is not abrupt. Since most of the enhanced field is
concentrated around the tip area, which accounts for .about.1/6 of
total area of the NPR, the actual molecule number contributing to
the Raman scattering signal in this high enhancement area is less
than 20, even if assuming the conjugation efficiency is 100% (FIG.
1d).
[0064] In a preferred embodiment, a positive control is used. For
example, in one embodiment, the intensities of the Raman peak for a
positive control as a function of PSA digestion time for various
PSA concentrations are obtained before detection of PSA presence or
activity in a sample. The typical SERS spectra of the
peptide-conjugated NPRs with positive controls biotin and R19 Raman
tag molecules are shown in FIGS. 3a and 3b, respectively. By
comparing the SERS spectra before and 2 hours after the peptide
digestion experiments, the Raman peaks from the NPR core (e.g.,
polystyrene core, e.g. 1003 cm.sup.-1) remain constant, and thus
can also serve as an internal control. The digestion rate is
related to the PSA concentration and PSA activity is typically
observed in 30 min for a concentration 1 nM (with .about.50%
reduction in biotin signal intensity, data not shown). Some Raman
peaks from the partial amino acid chain remaining on the NPR
surface after digestion may still appear in the spectra, although
the peak positions have slight changes and the peak intensities
decrease due to possible conformational changes upon peptide
cleavage.
[0065] In another embodiment, a negative control is run to show
that the peptides are specifically cleaved by protease present in
the sample. Example 1 shows the specificity of the conjugated
peptides to PSA using other serine proteases such as Granzyme B,
which can serve as a negative control. FIGS. 2B and 2C shows the
time-lapse SERS spectra of NPRs with R19 tag molecules in the two
control experiments with the PSA inhibitor and the serine protease
Granzyme B, which has orthogonal substrate specificity to PSA,
respectively. In the control experiment of peptide digestion by 420
nM Granzyme B, the reaction rate showed no statistically
significant difference from the inhibitor-treated reaction. The
inability for Granzyme B to cleave the peptide is also expected as
PSA has been shown to be the only protease for the HSSKLQ-LAAAC
sequence in vivo.
[0066] In a preferred embodiment, this peptide-conjugated NPR can
be used as a specific screening tool to provide information on the
concentration and proteolytic activity of the cancer biomarker PSA
in biological samples obtained from patients in a clinical
setting.
[0067] In another embodiment, the peptide-conjugated NPR can be
used as a specific screening tool to provide information on the
concentration and enzymatic activity of a biomarker, enzyme, kinase
or other protease in biological samples obtained from patients in a
clinical setting.
[0068] It is contemplated that one application of the present
probes is the incorporation of an NPR particle into a microfluidic
device which can automate and facilitate sample delivery and
washing process. The NPR particles can be also delivered in
real-time or immobilized in the device.
[0069] In another embodiment, NPRs can be spatially arranged in a
microarray format to achieve multiplexed measurements with broad
applications, by measuring all known proteases in unprocessed
biological samples without complex sample processing and
purification steps. The multiplexity of the substrate peptide
spatially can be achieved with a microarrayer with industry
standard protocols, when combined with NPR nanoarray clusters
arranged in microarray format. Even if each of the 500+ proteases
are cross-interrogated by 10 different substrate peptides, the
array still has an easily manageable feature number of less than
ten thousand.
Example 1
Raman Enhancement Factor of a Single Tunable Nanoplasmonic
Resonator (TNPR)
[0070] The TNPRs were patterned on quartz substrates (HOYA Co.) by
electron beam lithography (EBL) (Leica Microsystems Nanowriter
Series EBL 100). A 30 nm thick indium tin oxide (ITO) layer was
first sputtered on the substrate to prevent charging effects during
the EBL process. Poly(methyl methacrylate) (100 nm thick, MicroChem
Corp. PMMA) films, spincoated on the ITO-quartz glass, were used as
a positive photoresist. After exposure, the patterns were developed
using a 1:3 ratio of a methyl isobutyl ketone and isopropyl alcohol
mixture, followed by multilayer electron beam evaporation of silver
and oxide and standard lift-off procedures. We fabricated
three-layered Ag/SiO.sub.2/Ag and four-layered
Ag/SiO.sub.2/Ag/SiO.sub.2 TNPRs on the same substrate (we refer to
the four-layered TNPR as the capped TNPR in this example), with
each silver and SiO.sub.2 layer thickness fixed at 20 and 5 nm,
respectively. A shadow mask was inserted over the three-layered
TNPRs during deposition of the additional 5 nm SiO.sub.2 capping
layer to prevent deposition on the three-layered TNPRs. The samples
were measured by scanning electron microscopy and atomic force
microscopy to determine their sizes, shapes, and thicknesses. The
SiO2 layer sandwiched between the metallic layers can be used to
tune the surface plasmon resonance frequency by adjusting its
thickness. The EF can be maximized by matching the surface plasmon
resonance to the pump laser frequency. Under an optical microscope,
these particles, as shown in FIG. 1d, are distinctively visible due
to the strong scattering of light at resonant wavelengths. Scanning
electron microscopy (SEM) images (FIG. 1e) show that the TNPRs are
slightly elongated with a long axis and short axis of 117 and 81
nm, respectively. Atomic force microscopy (AFM) measurement (FIG.
1f) confirms that the height difference between capped TNPRs and
noncapped TNPRs is 5 nm, which matches the thickness of the SiO2
capping layer. Conducting AFM was used to ensure a good SiO.sub.2
layer coverage between the metal disks.
[0071] Scattering spectra were obtained by illuminating the TNPRs
with collimated light delivered by a multimode optical fiber from a
150 W Xe white light source. The light was delivered through a
right angle prism at an angle resulting in total internal
reflection (TIR), and the scattered light was collected with a JY
(550 grating) spectrometer system (Jobin Yvon). See FIG. 5B. For
the SERS experiment, SERS spectra were measured using a modified
Zeiss inverted confocal microscope with a 20.times. objective in a
backscattering geometry. An argon laser operating at 514 nm
(attenuated to -10 mW) was coupled into the microscope, and the
appropriate interference filters and holographic notch filter
(Kaiser) were placed in the beam path to remove the unwanted laser
lines. The output light path was coupled into the same spectroscope
as the scattering spectra measurement. See FIG. 5A.
[0072] In the measurements, the polarization of the incident
E-field is parallel to the TNPR long axis. FIG. 2a depicts the
scattering spectra of an individual TNPR and a capped TNPR with
resonant peaks at 516 and 536 nm, respectively. The sandwiched
SiO.sub.2 layer works as a tunable coupling factor for the plasmon
resonant frequency, and the top SiO.sub.2 layer works as a cover
layer to prevent the molecules from self-assembling on top of the
metal surface. The observed red-shift of the resonance spectrum of
the capped TNPR is due to the presence of the SiO.sub.2 layer,
which locally increases the refractive index.17 p-Mercaptoaniline
(pMA), a commonly used Raman dye, was selected in this study. The
low fluorescence emission background makes it a good candidate for
qualitative analysis of the Raman signal.
[0073] To ensure maximum coverage of the pMA monolayer on the TNPR
surface, the substrates were incubated in a 100 iM pMA solution for
more than 24 h. After repeatedly rinsing with DI water to remove
unwanted pMA adsorbed on the substrate and SiO.sub.2 capping layer,
the substrate was thoroughly dried with N2 gas. The spectra
measurements indicate the scattering peak positions of pMA-coated
capped and noncapped TNPRs were shifted to 534 and 550 nm,
respectively (FIG. 2b). The results indicate that the presence of
pMA in the vicinity of the TNPR causes a red-shift of the resonance
spectrum, equaling 18 nm for noncapped TNPRs and 14 nm for capped
TNPRs. This observation shows that pMA on top of an uncapped TNPR
does not lead to a significant shift (below 5 nm) and the surface
plasmon modes in the TNPR may be more sensitive to the local
refractive index change on the sidewall (in the direction of the
excitation electric field) than on the top surface. Numerical
simulations are more desirable to obtain a qualitative explanation
of the peak shift for different sample configurations. It should be
noted that these peak shifts suggest TNPRs can also serve as
molecule/bio sensors to monitor local refractive index changes.
[0074] The SERS EF for the TNPRs was evaluated by directly
comparing the SERS intensity obtained from TNPRs with unenhanced
molecules using the expression EF) (RS.sup.TNPR/RS.sup.reference)
([reference]/[TNPR]). RS.sup.TNPR and RS.sup.reference are the
measured SERS intensity of the TNPRs and normal Raman standard
sample, respectively. [TNPR] and [reference] are the estimated
number of molecules in the experiments. A saturated pMA monolayer
coverage of 0.39 nm2 per molecule is used to estimate the number of
molecules on a TNPR surface.18 Assuming that an individual TNPR is
a cylindrical ellipsoid particle, the maximum number of pMA
molecules that can self-assemble is .about.5.1.times.10.sup.4. A
0.1 mL neat liquid pMA (1.06 g/cm.sup.3) droplet on a quartz
substrate with a known detection volume from a 20.times. microscope
objective was used to estimate the number of molecules for the
reference sample, giving .about.4.5.times.10.sup.13 pMA molecules.
We took SERS spectra on a single TNPR at 10 different locations
with exactly the same exposure time and individual Raman spectra,
which are depicted in FIG. 3. Remarkably, the intensities of the
measured Raman spectra are very constant, showing that fabrication
repeatability of particles shapes is very reliable. Especially, the
signal strengths of the 1590 and 1077 cm-1 ring modes of pMA were
monitored. By looking carefully at the raw data, we found that the
intensity of these two modes is very constant among measurements.
The maximum SERS EF from an individual TNPR reached
.about.(3.4.+-.0.3).times.10.sup.10 and
(2.9.+-.0.3).times.10.sup.10 at 1077 and 1590 cm-1, respectively,
and the error bars were obtained from sample-to-sample variation of
Raman scattering intensity. It should be noted that the possible
error in estimating the surface coverage of pMA and the volume
illuminated by the microscope objective obviously affects the
precision in calculating the SERS EF. To provide a trustworthy
estimation of the SERS EF for a TNPR, we are performing the
calculation in the most conservative manner. The SERS EF was
determined by assuming maximum pMA coverage over the entire TNPR
metal surface and with the smallest volume being illuminated by
microscope objectives. The reported value is in fact representing
the lower bound of the SERS EF, and the actual EF can be even
higher.
[0075] We also measured the Raman spectra on SiO.sub.2-capped TNPRs
to isolate the SERS contribution from the sidewall of the capped
TNPRs. We assumed that there are no pMA molecules self-assembled on
the SiO2 layer after copious DI water rinsing. The ratio of pMA
molecules self-assembled on the exposed metal surface of the TNPR
to that of the capped TNPR was assumed to be the same as the area
ratio, which is about 1.6. The maximum measured SERS EF intensity
on the capped TNPR reaches (6.1.+-.0.3).times.10.sup.10 and
(4.6.+-.0.3).times.10.sup.10 for the 1077 and 1590 cm.sup.-1 ring
modes, respectively. The relative SERS intensity ratio for a TNPR
to a capped TNPR is approximately 1.3. This is an interesting
observation, since the SERS EF ratio between the capped TNPR and
the TNPR is not proportional to the number of molecules assembled
on the exposed metal surface.
[0076] To understand these experimental results, we performed
several numerical calculations. We applied the discrete dipole
approximation (DDA) method to compute the near field distribution
of the E-field surrounding the TNPR. FIG. 4 shows the calculated
E-field amplitude distribution at the sidewall and top surface of a
TNPR with the geometrical parameters corresponding to the SEM and
AFM measurements. The permittivity of Ag is taken from the
literature for bulk Ag material, and the permittivity of SiO.sub.2
is set to 2.13. The substrate, which has significant effects on the
plasmon resonance, is taken into account by embedding the particle
in a homogeneous medium with a refractive index of 1.4, which is
the averaged refractive index of air and ITO.26 FIG. 6c clearly
shows an angular dipole-like E-field distribution where stronger
fields are localized along the incident polarization direction
.theta.=0 (along the TNPR long axis). The z dependence of the field
shows that the strong local fields are mainly distributed close to
the Ag/SiO.sub.2, Ag/air, and Ag/ITO interfaces. FIG. 6b shows that
the stronger fields or "hotter spots" are mainly located close to
the edge of the ellipse around the direction of the incident
polarization.
[0077] In summary, we studied the SERS EF of TNPRs. The observed
SERS EF of a single TNPR can be as large as 6.1.times.10.sup.10
when the plasmon resonance is tuned to the pump laser frequency,
which is among the highest reported to date. We developed a novel
technique that forces the molecules to be assembled on the sidewall
of the resonator where the field is strongest, giving an accurate
measurement of the Raman enhancement factor. The experimental
results agree well with numerical calculations of the TNPR.
Nanofabrication enables precise dimension control and accurate
placement of the TNPRs, eliminating the issues resulting from
aggregation and size variation effects, often associated with
chemical synthesis or self-assembly of colloidal nanoparticles.
Thus, it offers a unique advantage for the development of
integrated biosensing devices.
Example 2
Protease-Specific Substrate Peptide Conjugated NPRs for Real-Time
Protease Detection and Measurement
[0078] In this work, NPRs (FIG. 1a) were conjugated with a PSA
protease-specific substrate peptide, which has the sequence
R19-HSSKLQLAAAC (SEQ ID NO:1) (S. R. Denmeade, C. M. Jakobsen, S.
Janssen et al., J Natl Cancer Inst 95 (13), 990 (2003); S. R.
Denmeade, W. Lou, J. Lovgren et al., Cancer Res 57 (21), 4924
(1997)), with the SERS molecule Rhodamine 19 (R19) at the
N-terminus and cysteine at the C-terminus (FIG. 1b). The peptide
has been identified as a highly specific peptides that can be
cleaved by paPSA in vivo in xenografts models (S. R. Denmeade, C.
M. Jakobsen, S. Janssen et al., J Natl Cancer Inst 95 (13), 990
(2003)) and human samples (P. Wu, U. H. Stenman, M. Pakkala et al.,
Prostate 58 (4), 345 (2004); P. Wu, L. Zhu, U. H. Stenman et al.,
Clin Chem 50 (1), 125 (2004)). The paPSA cleaves the peptide,
leading to the release of the R19 moiety (FIG. 1c) and a subsequent
decrease in the Raman scattering intensity in a dose- and
time-dependent manner, and the PSA protease activity can be
accurately quantified. It has been theoretically estimated that
SERS enhancement is strongly localized to the vicinity of
nanoparticle resonator surface (5-10 nm), which effectively
eliminates the assay background noise from the Raman scattering
substance in the surrounding fluids, or the R19 moieties that
diffuse into the solution after protease cleavage. Because of this
unique property, the assay can be performed in a simplified
one-step format with no additional washing step required.
[0079] Under an optical microscope, the NPR arrays were distinctly
visible due to the strong scattering of light at their resonant
wavelength (FIG. 1d). The magnified views of NPR arrays that
measured by Scanning Electron Microscope (SEM) and Atomic Force
Microscope (AFM) are showing in FIGS. 1e and 1f. The optical
properties of the NPR were characterized by illuminating the NPRs
with collimated light delivered by a multimode optical fiber from a
150 W Xenon lamp (Thermo Oriel) and collecting the extinction
spectra using a grating spectrometer (Triax 550, Jobin Yvon) with
matched liquid nitrogen cooled CCD detector (CCD-3500, Jobin Yvon)
(Durant S. Su K, Steel M. J., Xiong Y. Sun C., Zhang X, Journal of
Physical Chemistry B 110 (9), 3964 (2006)). The SiO.sub.2 layer,
sandwiched between the Ag layers, enable precisely tuning of NPR
resonance. As shown in FIG. 1g, the measured resonance peak of
NPRs-peptide-R19 conjugates closely matches laser excitation
wavelength at 532 nm, and thus, maximizes the enhancement of Raman
scattering. For the SERS experiments, Raman spectra were measured
using a modified inverted microscope (Axiovert 200, Zeiss) with a
50.times. objective in a backscattering configuration. As shown in
FIG. 1h, the NPR-based SERS substrate exhibits reproducible Raman
spectrum with consistent enhancement factor at the same order of
magnitude. The variation of experimentally measured SERS
intensities obtained from 6 different SERS NPR array are below 25%
and it can be normalized in the experiment.
[0080] The assay was performed by exposing the NPR-peptide
nanosensor to the fluidic samples and the subsequent time-dependent
R19 Raman spectra change was recorded at an interval of one minute
and an integration time of 30 seconds. The Raman 1316 cm.sup.-1
ring breathing mode from the PSA peptide was monitored as the
primary signature peak in this study, while the 1456 cm.sup.-1,
1526 cm.sup.-1, and 1597 cm.sup.-1 peaks were also monitored as
additional references (FIG. 2). The time-resolved spectral
measurement in the presence of PSA (FIG. 2a) is plotted in FIG. 2c.
Considering that the SERS intensities are proportional to the
remaining R19 on the NPR surface, the normalized SERS intensity
change is a direct indicator of PSA activity. Despite the variation
in the initial intensity of different Raman signature peaks, the
normalized data all converged into the same curve, indicating the
normalized SERS intensity change is a reliable measure to
quantitatively determine the substrate peptide being cleaved. As
shown in FIG. 2c, before PSA addition, the NPR nanosensor showed
steady intensities for all the peaks over a 10 minute period.
However, upon addition of PSA, a significant decrease in each Raman
peak is observed in the first 10-12 minutes, indicating that the
PSA protease was able to cleave the peptides on the NPR. The
decrease of the Raman signal then reaches a plateau by 30 minutes.
Another serine protease, Granzyme B, was selected as a negative
control (FIG. 2b-c). Within 50 minutes of recording, no substantial
changes in SERS intensity was observed, even at concentrations up
to 1 .mu.M. This result demonstrates that the decrease of Raman
signal in the PSA assay was based on a genuine enzymatic process,
rather than a non-specific hydrolysis reaction or due to the
displacement of the peptide from the surface by other components in
the buffer.
[0081] The sensitivity of the NPR nanosensor was evaluated by
measuring SERS intensity change of set of sample with PSA enzyme
concentration ranging from 6 nM to 6 pM. The absolute value of
normalized SERS intensity change is shown in FIG. 3a. As expected,
the rate of decrease in the Raman signal was proportional to the
concentration of PSA, which is explained by a reduced rate of
peptide cleavage when the PSA concentrations decreased. The
proteolytic activity eventually reaches an equilibrium stage at 30
minutes as indicated by each Raman signal reaching a plateau. The
absolute value of normalized decrease in SERS intensity versus
various concentrations, after 30 minutes of enzyme addition, is
plotted in FIG. 3b. The dynamic range for detection, in the current
assay setup, was from 6 pM to 6 nM. PSA concentration higher than 6
nM does not exhibit a distinct difference with the given detection
time. As shown in FIG. 3c, the monitored Raman peak intensity
exhibits distinguishable decay characteristics at different PSA
concentrations during the initial 4 minutes of sampling. Thus, by
fitting the decaying rate, reliable assays can possibly be
accomplished in 4 minutes.
[0082] In addition to protease activity measurements of purified
PSA, measurements for PSA protease activity in extracellular fluid
(ECF) from live cell culture was performed (FIG. 3d). It is well
known that LNCaP cells secrete PSA and have recently been used in
xenografts to evaluate in vivo PSA concentration (S. R. Denmeade,
C. M. Jakobsen, S. Janssen et al., J Natl Cancer Inst 95 (13), 990
(2003)). For comparison, a K562 cell line, which does not secrete
PSA into the ECF, was used as a negative control. LNCaP ECF showed
a significant change in SERS signal, while K562 exhibits very low
PSA enzymatic activity (FIG. 3d). By correlating the normalized
decrease in Raman signal with FIG. 3b, it was determined that the
LNCaP media had an elevated amount of paPSA concentration (FIG.
3d).
[0083] Compared with fluorescence-based assay, the NPR-based method
offers several advantages. Strongly localized Raman enhancement can
substantially amplify the signal and also effectively reduce
background noise. Therefore, it allows one-step and label free
detection of protease activity with sensitivity at 6 pM, and
dynamic range of 3 orders of magnitude. It should note PSA is
considered a weak protease and other proteases would allow even
better sensitivity. Second, it allows accurate measurement with
very small sample volume. Indeed, we estimate PSA protease activity
from single cells can be measured accurately (See below). Third,
fabricated using well established nano-lithography process,
NPR-based method is highly reproducible and thus, allowing
quantitative assessment of protease activity.
Methods
NPR Fabrication
[0084] The NPR was patterned on quartz substrates (HOYA Corp.) by
electron beam lithography (EBL) (Nanowriter Series EBL 100, Leica
Microsystems). A 30 nm thick indium-tin-oxide (ITO) under-layer was
sputtered on the substrate to prevent charging effects during the
EBL process. 100 nm-thick polymathylmethacrylate (PMMA, MicroChem
Corp.) films spin-coated on the ITO-quartz glass was used as a
positive photoresist. After exposure, the patterns were developed
using a 1:3 ratio of MIBK and IPA mixture followed by multilayer
deposition of metal and dielectric materials using electron beam
evaporation (Mark 40, CHA) and standard lift-off procedures. We
fabricated three layered Ag/SiO.sub.2/Ag NPR arrays with each
silver and SiO.sub.2 layer thickness equal to 25 nm and 5 nm,
respectively. The geometry of the fabricated NPR was examined by
atomic force microscopy, and the total thickness of the NPR is
confirmed to be 55 nm.
Device Design and Characterization
[0085] Micro-region SERS measurements are performed on an inverted
optical microscope (Axiovert 200, Zeiss) with matched high
resolution grating spectrameter (Triax 550, Jobin Yvon).
Instrumental set-up is shown in FIGS. 5A and 5B. To excite the
particles plasmons, the Nanoplasmonic Resonators (NPRs) were
illuminated with a collimated laser beam (Frequency doubling YAG
laser, 532 nm) through a right angle prism at an angle resulting in
a total internal reflection (TIR) configuration. An evanescent
electromagnetic wave is generated and used to excite the particle
plasmons. The samples are attached to the bottom of the prism by
applying index matching oil between the sample substrate and the
prism. Such sample configuration effectively eliminates all stray
scattering light due to surface defects and dust particles, leading
to significantly reduced background signal. The excited collective
electron oscillations within the particles then radiate
electromagnetic waves of the same frequency into the far field,
whereby the collection and spectral measurement takes place. The
scattered light from the NPRs is then collected by a 50.times. long
working distance objective. A holographic notch filter (532 nm,
Kaiser Optical System) was placed in the beam path in order to
remove the illuminating beam. The emerging light is then imaged
onto the entrance slit of the grating spectrometer system with a
liquid Nitrogen cooled charge coupled device (CCD) camera
(CCD-3500, Jobin Yvon) for spectrum analysis.
[0086] The extinction spectrum measurement was performed in a
similar configuration. In this measurement, 150 W Xenon white light
source has been used as the illumination source and the collimated
light was delivered through an optical fiber bundle.
[0087] For the SERS experiments, Raman spectra were measured using
a modified inverted microscope (Axiovert 200, Zeiss) with a
50.times. objective in a backscattering configuration. Baseline
subtraction was applied to remove the fluorescence background of
the measured spectra. The spectra were then smoothed in Matlab
using the Savitsky-Golay method with a second-order polynomial and
window size of 9. To correct the possible influence due to the
fluctuation of illumination intensity, frequency dependence of
Raman scattering, and the variation of initial packing density of
the report molecules, the change in SERS intensity was normalized
to the average intensity before protease addition. The normalized
SERS intensity change is defined as
.DELTA.I=[I.sub.t-I.sub.0]/I.sub.0
where I.sub.0 is the average SERS intensity before the addition of
the protease and I.sub.t is the SERS intensity measured at the
given time t.
[0088] To estimate the detection volume of the NPR array, the
diffusion length is first calculated as L.sub.D= {square root over
(Dt)} .about.0.5 mm where D is estimated as 1.times.10.sup.-6
cm.sup.2/s and t is 1800 s. The un-normalized detection volume is
then determined as V=(L.sub.A+L.sub.D).sup.2 L.sub.D
.about.1.times.10.sup.-7 L where L.sub.A is the length of the NPR
array (15 .mu.m). This volume must be normalized due to the fact
that the molecules have an equal likelihood to diffusion in any
direction. The likelihood of these molecules coming in contact with
the NPR array can be estimated based on the surface area of the
array and the total surface area available for diffusion. This
probability of molecules can diffuse to the NPR array is then given
by:
W Det = L A 2 6 L D 2 .about. 1.5 .times. 10 - 4 ##EQU00001##
assuming the NPR array surface area of the array is much smaller
than the available diffusion surface area. The total sampling
volume is then calculated as V.sub.Det=W.sub.DetV .about.15 pL.
Peptide Synthesis
[0089] R6G-Ava-HSSKLQLAAAC-NH.sub.2 (SEQ ID NO:1). (2). 401 mg
(0.277 mmol) of Rink Amide AM polystyrene resin (loading 0.69
mmol/g) was added to a 12 mL fitted syringe and swollen with NMP (4
mL). The Fmoc protecting group was removed by treatment with 1:2:2
piperidine/NMP/CH.sub.2Cl.sub.2 solution (3 mL) for 30 min, and the
resin was filtered and washed with NMP (3.times.3 mL) and
CH.sub.2Cl.sub.2 (3.times.3 mL). To load the .alpha.-amino acid
residues, the resin was subjected to repeated cycles of coupling
conditions (method A or method B), followed by washing (5.times.3
mL NMP, 5.times.3 mL CH.sub.2Cl.sub.2), Fmoc deprotection
[treatment with 1:2:2 piperidine/NMP/CH.sub.2Cl.sub.2 solution (3
mL) for 30 min], and washing again with NMP (5.times.3 mL) and
CH.sub.2Cl.sub.2 (5.times.3 mL). The first .alpha.-amino acid
residue was loaded by addition of a preformed solution of
Fmoc-Cys(Trt)-OH (1.17 g, 2.00 mmol), PyBOP (1.04 g, 2.00 mmol),
and HOBt (270 mg, 2.00 mmol) in 1:1 NMP/CH.sub.2Cl.sub.2 (2 mL)
onto the resin and the resulting slurry was stirred for 5 min on a
wrist-action shaker, followed by addition of i-Pr.sub.2EtN (0.55
mL, 4.0 mmol). The reaction was allowed to proceed for 5 h. The
resin was then filtered, washed (5.times.3 mL NMP, 5.times.3 mL
CH.sub.2Cl.sub.2), and dried under high vacuum. The loading of Cys
was determined to be 0.60 mmol/g (78% yield). Successive couplings
were achieved either by method A or method B. Method A consists of
addition a preformed solution of Fmoc-protected amino acid
[Fmoc-Cys(Trt)-OH (1.17 g, 2.00 mmol), Fmoc-Ala-OH (622 mg, 2.00
mmol), Fmoc-Leu-OH (707 mg, 2.00 mmol), Fmoc-Gln(Trt)-OH (1.22 g,
2.00 mmol), Fmoc-Ser(tBu)-OH (767 mg, 2.00 mmol), and
Fmoc-His(Trt)-OH (1.24 g, 2.00 mmol)], PyBOP (1.04 g, 2.00 mmol),
and HOBt (270 mg, 2.00 mmol) in NMP/CH.sub.2Cl.sub.2 (1:1, 2 mL),
followed by addition of i-Pr.sub.2EtN (0.55 mL, 4.0 mmol). The
reactions were allowed to proceed for at least 4 h. Method B
consists of subjection of the resin to a 0.4 M solution of the
suitably protected acid [Fmoc-Lys(Boc)-OH (375 mg)], which had been
pre-activated by incubation with DIC (130 .mu.L, 0.84 mmol) and
HOBt (108 mg, 0.800 mmol) in DMF (2 mL) for 10 min. The coupling
was allowed to proceed for 4 h. After each coupling the resin was
filtered and washed (NMP: 5.times.3 mL, CH.sub.2Cl.sub.2: 5.times.3
mL), followed by removal of the Fmoc protecting group. After
coupling and deprotection of the final .alpha.-amino acid residue,
the Ava linker was added by subjection of the resin to a 0.4 M
solution of Fmoc-S-Ava-OH (272 mg, 0.800 mmol) which had been
pre-activated by incubation with DIC (120 .mu.L, 0.80 mmol) and
HOBt (108 mg, 0.800 mmol) in N-methylpyrrolydinone (1 mL) for 10
min. The coupling was allowed to proceed overnight. The resin was
filtered and washed (5.times.3 mL NMP, 5.times.3 mL
CH.sub.2Cl.sub.2), the Fmoc protecting group was removed, and the
resin washed again. The rhodamine group was incorporated by adding
a 0.4 M solution of rhodamine 19 (412 mg, 0.8 mmol), which had been
pre-activated by incubation with DIC (130 .mu.L, 0.84 mmol) and
HOBt (108 mg, 0.800 mmol) in NMP (2 mL) for 10 min. The reaction
was allowed to proceed for 6 h, the coupling procedure was repeated
once more and the reaction was allowed to proceed overnight. The
substrate was cleaved from the resin by incubation with a solution
of 94:2:2:2 TFA/triisopropylsilane/H.sub.2O/ethanedithiol (3 mL)
for 2 h, purified using preparatory C18 reverse-phase HPLC
(CH.sub.3CN/H.sub.2O-0.1% TFA, 5-95% for 50 min, 20 mL/min,
220/254/280 nm detection for 100 min, t.sub.R=24.3 min), and
lyophilized. MS (MALDI), m/z calcd for
C.sub.78H.sub.116N.sub.19O.sub.175: 1622.85. Found: m/z
1623.90.
Peptide Conjugation to NPR
[0090] The peptide attaches to the NPR via the thiol group on
cysteine. The PSA substrate peptides mixed with octanethiol at a
1:3 ratio were incubated for 24 hours to ensure a well-ordered
self-assembled monolayer (SAM) on the NPR metallic surface.
Octanethiol, with a SAM chain length of 1-2 nm, was used as packing
material to manage the distance among the PSA substrate peptides
and help to erect the PSA substrate peptide for optimal spatial
presentation. Thus, the octanethiol SAM improves access for the
protease to bind to the peptides.
Cell Culture and Clinical Semen Samples
[0091] LNCaP cells actively secrete PSA into ECF, and the human CML
cells K562 are negative for PSA. Both cell lines are maintained in
RPMI-1640 with 10% FBS and 1.times. Pen/Strep, at 37.degree. C.
with 5% CO.sub.2. 10.times.10.sup.6 cells were cultured overnight
in 10 ml fresh media. Media from both cultures were collected and
PSA activity was measured by fluorescent methods as described
before, and calibrated against commercial PSA (Calbiochem, San
Diego, Calif.). Briefly, the PSA-binding peptides and derivatives
with a spacer were chemically synthesized and used to prepare an
affinity column, which was used to fractionate PSA in seminal
plasma.
Purified PSA Preparation and Proteolytic Reaction
[0092] Proteolytically active PSA was purified to homogeneity from
human seminal plasma by column chromatography, eliminating all
known PSA complexes and retaining its protease fraction. Cleavage
of the substrate peptide immobilized on the NPR nanosensor is
performed in a buffer of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, and
0.1 mM EDTA, and the reaction was monitored in real-time in
37.degree. C. Protease inhibitors (to prevent PSA and Granzyme B
degradation) are obtained from CalBiochem and added to the reaction
following the manufacturer's instructions, so that the final
reaction solution contains 5 .mu.M AEBSF, 4.2 nM Aprotinin, 200 nM
Elastatinal and 10 nM GGACK. The concentration of proteolytically
active PSA in the PSA reagent has been prepared with a wide range
of concentration from 6 pM to 6 nM.
[0093] The present examples, methods, procedures, specific
compounds and molecules are meant to exemplify and illustrate the
invention and should in no way be seen as limiting the scope of the
invention. Any patents, publications, publicly available sequences
mentioned in this specification and below are indicative of levels
of those skilled in the art to which the invention pertains and are
hereby incorporated by reference to the same extent as if each was
specifically and individually incorporated by reference.
Sequence CWU 1
1
17111PRTArtificialSynthetic PSA-specific peptide substrate 1His Ser
Ser Lys Leu Gln Leu Ala Ala Ala Cys 1 5 10 27PRTArtificialSynthetic
peptide substrate of cAMP-dependent protein kinase 2Leu Arg Arg Ala
Ser Leu Gly 1 5 39PRTArtificialSynthetic peptide substrate of cAMP
dependent protein kinase (PKA) 3Gly Arg Thr Gly Arg Arg Asn Ser Ile
1 5 411PRTArtificialSynthetic peptide substrate of protein kinase C
(PKC) 4Gln Lys Arg Pro Ser Gln Arg Ser Lys Tyr Leu 1 5 10
57PRTArtificialSynthetic peptide substrate of protein kinase
Akt/PKB 5Arg Pro Arg Ala Ala Thr Phe 1 5 612PRTArtificialSynthetic
peptide substrate of Abl kinase 6Glu Ala Ile Tyr Ala Ala Pro Phe
Ala Lys Lys Lys 1 5 10 715PRTArtificialSynthetic peptide substrate
of 5prime-AMP-activated protein kinase (AMPK) 7His Met Arg Ser Ala
Met Ser Gly Leu His Leu Val Lys Arg Arg 1 5 10 15
813PRTArtificialSynthetic peptide substrate of
Ca2+/calmodulin-dependent protein kinase 8Lys Lys Ala Leu Arg Arg
Gln Glu Thr Val Asp Ala Leu 1 5 10 98PRTArtificialSynthetic Peptide
substrate of cyclin-dependent kinase 2 (cdc2) 9Ser Pro Gly Arg Arg
Arg Arg Lys 1 5 107PRTArtificialSynthetic peptide substrate of
cyclin-dependent kinase 2 (Cdk2) 10His His Ala Ser Pro Arg Lys 1 5
1110PRTArtificialSynthetic peptide substrate of cyclin-dependent
kinase 5 (Cdk5) 11Pro Lys Thr Pro Lys Lys Ala Lys Lys Leu 1 5 10
1218PRTArtificialSynthetic peptide substrate of casein kinase 1
(CK1) 12Arg Arg Lys Asp Leu His Asp Asp Glu Glu Asp Glu Ala Met Ser
Ile 1 5 10 15 Thr Ala 1310PRTArtificialSynthetic peptide substrate
of CK2 alpha subunit or holoenzyme 13Arg Arg Arg Asp Asp Asp Ser
Asp Asp Asp 1 5 10 1414PRTArtificialSynthetic peptide substrate of
DYRK family protein kinases 14Lys Lys Ile Ser Gly Arg Leu Ser Pro
Ile Met Thr Glu Gln 1 5 10 1526PRTArtificialSynthetic peptide
substrate of GSK3 alpha and beta kinases 15Tyr Arg Arg Ala Ala Val
Pro Pro Ser Pro Ser Leu Ser Arg His Ser 1 5 10 15 Ser Pro His Gln
Ser Glu Asp Glu Glu Glu 20 25 1615PRTArtificialSynthetic peptide
substrate of Src Kinase 16Lys Val Glu Lys Ile Gly Glu Gly Thr Tyr
Gly Val Val Tyr Lys 1 5 10 15 1723PRTArtificialSynthetic peptide
substrate of checkpoint kinases CHK1 and CHK2 17Lys Lys Lys Val Ser
Arg Ser Gly Leu Tyr Arg Ser Pro Ser Met Pro 1 5 10 15 Glu Asn Leu
Asn Arg Pro Arg 20
* * * * *