U.S. patent application number 11/637831 was filed with the patent office on 2008-06-19 for incorporation of biomolecules in thin films.
Invention is credited to Peter J. Edmonson, Dennis William Hess, William D. Hunt, Sang Hun Lee, Prabhakar Apparao Tamirisa.
Application Number | 20080142366 11/637831 |
Document ID | / |
Family ID | 39525826 |
Filed Date | 2008-06-19 |
United States Patent
Application |
20080142366 |
Kind Code |
A1 |
Tamirisa; Prabhakar Apparao ;
et al. |
June 19, 2008 |
Incorporation of biomolecules in thin films
Abstract
A method of incorporating biomolecules in a thin film mounted on
a substrate, with the film having a thickness of not more than
about 10 microns, includes providing a metal structure on the
substrate between the thin film and the substrate, positioning a
medium containing biomolecules in contact with a side of the film
remote from the metal substrate, and applying a predetermined
electrical voltage between the metal substrate and the medium to
cause biomolecules to migrate in an electrophoretic manner from the
medium into the thin film.
Inventors: |
Tamirisa; Prabhakar Apparao;
(Atlanta, GA) ; Lee; Sang Hun; (Yong-In, KR)
; Hunt; William D.; (Decatur, GA) ; Hess; Dennis
William; (Atlanta, GA) ; Edmonson; Peter J.;
(Hamilton, CA) |
Correspondence
Address: |
GOWLING, LAFLEUR HENDERSON LLP
ONE MAIN STREET WEST
HAMILTON
ON
L8P 4Z5
omitted
|
Family ID: |
39525826 |
Appl. No.: |
11/637831 |
Filed: |
December 13, 2006 |
Current U.S.
Class: |
204/456 |
Current CPC
Class: |
C25D 13/20 20130101;
C25D 13/04 20130101 |
Class at
Publication: |
204/456 |
International
Class: |
C25D 15/00 20060101
C25D015/00 |
Claims
1. A method of incorporating biomolecules in a thin film mounted on
a substrate, with the film having a thickness of not more than
about 10 microns, said method including: providing a metal
structure on the substrate between the thin film and the substrate,
positioning a medium containing biomolecules in contact with a side
of the film remote from the metal substrate, and applying a
predetermined electrical voltage between the metal substrate and
the medium to cause biomolecules to migrate in an electrophoretic
manner from the medium into the thin film.
2. A method according to claim 1 wherein the biomolecules comprise
molecular recognition elements.
3. A method according to claim 2 wherein the molecular recognition
elements comprise antibodies.
4. A method according to claim 1 including positioning spaced apart
first and second metal structures on the substrate between the film
and the substrate, and applying a predetermined electrical voltage
between the first metal structure and the medium and applying a
different predetermined electrical voltage between the second metal
structure and the medium to cause different migration of
biomolecules from the medium into first and second portions of the
thin film adjacent the first and second metal structures
respectively.
5. A method according to claim 1 wherein the substrate comprises a
piezoelectric material and the metal structure and substrate are
portions of an acoustic wave device.
6. A method according to claim 1 wherein the thin film is a
hydrogel:
7. A method according to claim 1 wherein the thin film is a
hydrogel, the biomolecules comprise antibodies and the biomolecule
containing medium comprises a buffer solution.
Description
FIELD OF INVENTION
[0001] The present invention relates to the incorporation of
biomolecules in a thin film, namely a film with a thickness of not
more than about 10 microns.
BACKGROUND OF THE INVENTION
[0002] Various methods of incorporating biomolecules in thin films
have previously been proposed, for example a plasma polymerization
method and a spin casting method, but none have been particularly
satisfactory. The plasma polymerization method and the spin casting
method will be commented on in more detail later.
[0003] It is an object of this invention to provide an improved
method for incorporating biomolecules in thin films.
SUMMARY OF THE INVENTION
[0004] The present invention provides a method of incorporating
biomolecules in a thin film mounted on a substrate, with the film
having a thickness of not more than about 10 microns, said method
including providing a metal structure on the substrate between the
thin film and the substrate, positioning a medium containing
biomolecules in contact with a side of the film remote from the
metal substrate, and applying a predetermined electrical voltage
between the metal substrate and the medium to cause biomolecules to
migrate in an electrophoretic manner from the medium into the thin
film.
[0005] The biomolecules may comprise molecular recognition elements
(MREs), and the molecular recognition elements may comprise
antibodies.
[0006] The method may include positioning spaced apart first and
second metal structures on the substrate between the film and the
substrate, and applying a predetermined electrical voltage between
the first metal structure and the medium and applying a different
predetermined electrical voltage between the second metal structure
and the medium to cause different migration of biomolecules from
the medium into first and second portions of the thin film adjacent
the first and second metal structures respectively.
[0007] The substrate may be of piezoelectric material and the metal
structure and substrate are portions of an acoustic wave device.
The film may comprise a hydrogel. The thin film may comprise a
hydrogel, the biomolecules may comprise antibodies and the
biomolecule containing medium may comprise a buffer solution.
[0008] According to one aspect of the present invention, a method
of incorporating protein biomolecules, such as antibodies and
enzymes, nucleic acids (DNA and RNA), and other molecular
recognition elements into thin films obtained through plasma
polymerisation for the production of acoustic wave biosensors
utilizes electrophoresis. The production of MRE-containing thin
films with such a method can be more readily controlled when
compared to production by previous methods. The metal structure of
the acoustic wave device on which an MRE-containing thin films is
located can be easily adapted to permit the formation of localized
electrodes which will confine the placement of different MREs to
certain areas within the acoustic wave structure during the
electrophoretic process.
[0009] The method of preparing thin films using electrophoresis in
accordance with this invention can also be extended to include
other devices which require their biomolecules not to diffuse out
of the film when-placed in a solution. The electrophoretic
technique promotes incorporation of biomolecules into the film by
use of an electric field and thereby accomplishes the correct
orientation of the biomolecules and prevents the biomolecules from
diffusing from the film in the absence of an electric field. A
method according to the present invention can be applied to various
optical, micro mechanical system (MEMS) and nano mechanical system
(NEMS) biosensors, and drug delivery systems used in medical
applications. Another use of modified thin films to which the
present invention can be applied is to alter the mechanical and
physical properties of the surface for MEMS and NEMS scale devices.
Such applications focus on the wetting, lubrication and protection
properties of the thin films.
[0010] Thus, a major aspect of this invention is to provide an
improvement in the placement and retention of biomolecules within
certain thin films using electrophoresis. The present invention can
also provide improvements in how the metal structure of an acoustic
wave device can be modified to easily implement the localized
placement of several different biomolecules using
electrophoresis.
[0011] This invention provides a method of preparing biomolecule
sensitive thin films using an electrophoretic technique for the
large scale manufacture of biosensors. Such biosensors can be
incorporated into various detection systems and configurations, for
example as described in:
[0012] U.S. Pat. No. 7,053,524 B2 issued May 30, 2006 to Edmonson
et al, and entitled "A SURFACE ACOUSTIC WAVE SENSOR OR
IDENTIFICATION DEVICE WITH BIOSENSING CAPABILITY".
[0013] U.S. patent application Ser. No. 11/088809, filed Mar. 25,
2005 by Edmonson et al, and entitled "DIFFERENTIATION AND
IDENTIFICATION OF ANALOGOUS CHEMICAL OR BIOLOGICAL SUBSTANCES WITH
BIOSENSORS".
[0014] U.S. patent application Ser. No. 60/613262, filed Sep.
24,2004 by Stubbs et al, and entitled "SURFACE ACOUSTIC WAVE
IMMUNOSENSORS FOR THE DETECTION OF SIGNALING MOLECULES IN A
BIOLOGICAL ENVIRONMENT".
[0015] Other types of biosensors such as a thin film optical
biosensor described by Xiao-bo Zhong et al, "Single-nucleotide
polymorphism genotyping on optical thin-film biosensor chips,"
PNAS, Vol. 100, No. 20, pp. 11559-11564, Sep. 30, 2003 and Guoguang
Rong et al, "High Sensitivity Sensor Based on Porous Silicon
Waveguide," Materials Research Society Symp. Proc. Vol. 934, 2006
would benefit from this invention.
[0016] This invention would benefit other thin films produced by
various methods, such as polymerization, and other methods such as
spin casting as described by P: Cooreman et al, "Thin Polymer Films
as Substrates for Biosensor Applications," Institute for Materials
Research, Limburgs Universitair Centrum Wetenschapspark 1, 3590
Diepenbeek, Belgium.
[0017] This invention would improve other applications of
re-engineered thin films. For example, the modification of various
surfaces for medical implants, with protective biomolecule coatings
which are designed to enhance biocompatibility with surrounding
tissue. Another example is in the flexible electronics industry
which can utilize thin films which are embedded with functional
biomolecules such as ferritin. Another use of re-engineered thin
films is to modify the mechanical and physical properties of the
surfaces of MEMS and NEMS scale devices.
[0018] This invention may also be useful to develop chemical or
biomolecule containing thin films for applications in
micro-bio/chemical systems (microscale chemical systems analogous
to MEMS) such as microreactors. For example, enzymes incorporated
in hydrogel or polymer thin films may be used to catalyse
biochemical reactions.
[0019] This invention may also be useful to develop
protein/biomolecule arrays by "electrophoretic spotting" of
suitable carrier materials such as polymers, hydrogels or ceramics.
Protein/biomolecule arrays have applications in combinatorial
testing, drug discovery, and fundamental studies of biomolecule
interactions.
[0020] This invention also provides a method for producing an
acoustic wave biosensor using an electrophoretic procedure for
post-deposition of antibodies and other potentially charged MREs
from buffer solutions into customized hydrogel thin films produced
in a well-controlled and reproducible RF plasma polymerization
process. Electrophoretic incorporation of MREs in hydrogel films is
relatively simple to implement and can be used to control the
orientation of antibodies and other like molecules in the hydrogel.
An example which will be described in more detail later will show
how the negatively charged F.sub.c portion of an antibody is
attracted to a positively biased metal structure underlying a
hydrogel film derived from N-Isopropylacrylamide (NIPAAm), which
leaves the F.sub.ab portion to bind the antigen. Since
electrophoretic transfer permits incorporation of the antibodies
and other MREs into the hydrogel thin film network, higher
densities of molecular recognition centers can be achieved using
this technique than when the antibodies and other MREs are
covalently bonded to the surface of the hydrogel. Further, a
modification of the acoustic wave metal structure permits
controlled localized MRE placement by separately biasing the metal
structure areas under the polymer film.
[0021] Recently, there has been extensive activity in the
production of both vapor phase and liquid phase acoustic wave
biosensors for the detection of various substances ranging from
illicit drugs and explosives to harmful pathogens and cancer
biomarkers. The following publication list outlines the interests
currently being pursued in the acoustic wave biosensor area:
[0022] Christopher D. Corso, Desmond D. Stubbs, Sang-Hun Lee,
Michael Goggins, Ralph H. Hruban, and William D. Hunt, "Real-time
detection of mesothelin in pancreatic cancer cell line supernatant
using an acoustic wave immunosensor," Cancer Detection and
Prevention Journal, vol. 30, pp. 180-187, 2006.
[0023] Sang-Hun Lee, D. D. Stubbs, J. Cairney, and W. D. Hunt,
"Rapid Detection of Bacterial Spores Using a Quartz Crystal
Microbalance (QCM) Immunoassay," IEEE Sensors Journal, vol. 5, no.
4, pp. 737-743, 2005.
[0024] Desmond D. Stubbs, Sang-Hun Lee, and William D. Hunt, "Vapor
Phase Detection of a Narcotic Using Surface Acoustic Wave
Immunoassay Sensors," IEEE Sensors Journal, vol. 5, no. 3, pp.
335-339, 2005.
[0025] Sang-Hun Lee, D. D. Stubbs, W. D. Hunt, and P. J. Edmonson,
"Vapor Phase Detection of Plastic Explosives Using a SAW Resonator
Immunosensor Array," 2005 IEEE Sensors Conference, pp. 468-471,
Irvine, Calif., 2005.
[0026] L.A. Francis et al, "A SU-8 liquid cell for surface acoustic
wave biosensors," MEMS, MOEMS, and micromachining Conference,
Strasbourg , FRANCE vol. 5455, pp. 353-363, 2004.
[0027] W. D. Hunt, D. D. Stubbs and Sang-Hun Lee, "Time-Dependent
Signature of Acoustic Wave Biosensors," Proceedings of IEEE, vol.
91, pp 890-901, 2003.
[0028] K. Lange et al, "A Surface Acoustic Wave Biosensor Concept
with Low Flow Cell Volumes for Label-Free Detection," Anal. Chem.,
75 (20), 5561-5566, 2003.
[0029] G. Auner et al, "Dual-mode acoustic wave biosensors
microarrays," Bioengineered and Bioinspired Systems. Edited by
Rodriguez-Vazquez et al, Proceedings of the SPIE, Volume 5119, pp.
129-139, 2003.
[0030] F. Bender et al, "Love-wave biosensors using cross-linked
polymer waveguides on LiTaO substrates," Electronics Letters, Vol.
36, No. 19, 2000.
[0031] J. Freudenberg et al, "A contactless surface acoustic wave
biosensor," Biosensors and Bioelectronics, Elsevier Science, Vol.
14, No 4, pp. 423-425, 30 Apr. 1999.
[0032] The common necessity of the acoustic wave biosensors
mentioned in the above publications is the requirement that the
biomolecule immobilization on the acoustic wave biosensor surface
is both stable and repeatable. Biolayer parameters such as
thickness and biomolecule receptor density contribute to a stable,
repeatable sensing device.
DESCRIPTION OF THE DRAWINGS
[0033] Embodiments of the invention will now be described, by way
of example, with reference to the accompanying drawings, of
which:
[0034] FIG. 1 is a cross-sectional view of the basic structure of
an acoustic wave biosensor to which the present invention can be
applied,
[0035] FIG. 2 is a schematic view of prior art methods of
crosslinked hydrogel film deposition, namely a plasma
polymerisation method in FIG. 2(a) and a spin coating method in
[0036] FIG. 2(b),
[0037] FIG. 3 is a schematic view of an antibody structure and its
fragments,
[0038] FIG. 4 is a schematic of apparatus for carrying out a method
of electrophoretic immobilization on an acoustic wave device in
accordance with one embodiment of the invention,
[0039] FIG. 5 is a schematic of an AT-cut quartz crystal plate bulk
acoustic wave device to which the present invention can be
applied,
[0040] FIG. 6 is a schematic view of an acoustic wave delay line
structure to which the present invention can be applied,
[0041] FIG. 7 is a similar view of a SAW two-port resonator
structure to which the present invention can be applied,
[0042] FIG. 8 is a similar view of a SAW two-port resonator
structure with an energy trapping film to which the present
invention can be applied,
[0043] FIG. 9 is a similar view of a reflective type RFID/biosensor
with multiple reflector arrays to which the present invention can
be applied,
[0044] FIG. 10 is a similar view of an RFID/biosensor with
selectable EDT arrays to which the present invention can be
applied,
[0045] FIG. 11 is a similar view showing inter-IDT pad
connectivity,
[0046] FIG. 12 is a schematic view of an apparatus for carrying out
three-electrode electrophoretic immobilization of an acoustic wave
device in accordance with another embodiment of the invention,
[0047] FIG. 13 is a schematic view of a thin film structure to
which the present invention can be applied, and
[0048] FIG. 14 is a schematic view of apparatus for carrying out
multi-electrode eledtrophoretic immobilization on a thin film
structure.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0049] The acoustic wave is the dominant factor that determines
detection sensitivity of an acoustic wave biosensor. The acoustic
wave can be defined either as various derivations of bulk waves or
surface waves, as described by C. K. Campbell, Surface Acoustic
Wave Devices for Mobile and Wireless Communications, Academic
Press, 1998. In general, the acoustic wave propagates through
piezoelectric material and can generate changes in its properties
(phase shift, velocity change, attenuation, etc.) that are caused
by the interaction of the acoustic wave with target material
embedded within a selective biolayer. Such changes in acoustic wave
properties can be electrically transformed via the piezoelectric
effect and metal structure into a detectable electrical signal.
FIG. 1 is a cross-sectional view of the basic structure of an
acoustic wave biosensor 100. The piezoelectric material 110 is
chosen to support the required acoustic wave 115 to meet the
application objective of the biosensor. This piezoelectric material
110 may be in the form of a crystal substrate or a deposited
piezoelectric film.
A metal structure 120 is placed on the piezoelectric material 110
by any one of a number of thin film deposition techniques. The main
purpose of the metal structure 120 is to present an electric field
to the piezoelectric material so that an acoustic wave 115 can be
developed. Due to the reciprocal nature of the piezoelectric
material 110 and the metal structure 120, interaction between the
acoustic wave 115 with the metal structure 120 can subsequently
produce an electric field. A polymer thin film 130, such as a
hydrogel, is placed in contact with the metal structure 120 and/or
the bare surface of the piezoelectric material 110, depending on
the specific application of the biosensor. The thickness of the
polymer film, T.sub.P 135, is critical in determining
reproducibility of the sensitivity of the biosensor. Atibodies,
aptamers or other suitable MRE material 140 is then interfaced with
the polymer film 130 to form a receptor layer 140. A critical
parameter which affects the performance of the biosensor is the
density DA 145 of the MRE material which prescribes how many
antibodies or other suitable MRE material 140 reside within the
upper volume of the polymer thin film 130.
[0050] The present invention will show that, by improving the
parameters of the polymer thin film 130 and carrying out an
electrophoretic process to interface the antibodies and/or other
suitable MRE material 140 with the polymer thin film 130, an
improved biosensor can be produced.
[0051] Sensor performance is greatly affected by the nature of the
polymer film, which comprises a polymer film matrix containing
immobilized MREs. Traditionally, antibodies have been immobilized
in polyethyleneimine or (.gamma.-aminopropyl) triethoxysilane
matrices, as described by K. Nakanishi, et. al., "A novel method of
immobilizing antibodies on a quartz crystal microbalance using
plasma-polymerized films for immunosensors," Anal Chem, vol. 68,
pp. 1695-700, 1996. Prior art film formation methods such as spin
casting or dip coating can result in thick films and hence a large
mass on the surface of the acoustic wave device, which contributes
to instabilities in the properties of the piezoelectric material.
Plasma deposition of polymeric layers is a viable alternative to
conventional thin film fabrication methods for sensors. Plasma
polymerized films are pinhole free, conformal, can be integrated
with other dry, vacuum based microelectronic technologies and
eliminate the need for solvents, thus mitigating environmental
concerns. Further, ultra thin coatings. (2-20 nm) can be obtained
which are resistant to delamination due to their excellent adhesion
and do not deleteriously affect the mechanical properties of
piezoelectric material in acoustic wave sensors.
[0052] Hydrogels are water swollen polymeric networks which are
ideal for the creation of antibody-polymer and other like
MRE-polymer composites due to their many potential applications in
the field of biosensors. Current approaches to producing
MRE-sensitive hydrogel thin films include covalent and non-covalent
techniques. The covalent approach, which involves various
chemistries to conjugate the biomolecules to hydrogels and other
host materials is labor intensive and requires expensive reagents,
as outlined in Mayes, A. G. et. al., Immobilization chemistry,
Biomolecular sensors,: pp. 78, New York, N.Y., 2002. The
non-covalent approaches necessitate a thin film forming process
such as spin casting from bulk gels containing biomolecules as
described by O'Driscol, K. F., Techniques of enzyme entrapments in
gels. Academic Press: New York, 1976; Vol. XXXIV, p 169-243. Since
spin casting from complex bulk materials does not produce uniform,
homogeneous films of low thicknesses, the approach is not suitable
for use in high-sensitivity sensors, as mentioned in Avramov, I.
D., et. al. Investigations on plasma polymer coated SAW and STW
resonators for chemical gas sensing applications. IEEE Trans.
Microwave Theory Tech. 2001, 49, (4), 827-837.
[0053] Hydrogels are water-swollen crosslinked polymeric structures
derived from hydrophilic monomers. They are produced by the
polymerization of one or more monomers or by the association of
bonds such as hydrogen bonds and strong van der Waals interactions
between polymeric chains, as documented in N. A. Peppas, "Hydrogels
in medicine and pharmacy." Boca Raton, Fla.: CRC Press, 1987.
Appropriate crosslink densities are built into the structures
either during polymerization by incorporating free radical
crosslinking agents, or by post-gel formation exposure to
radiation, as explained in A. Chapiro, Radiation chemistry of
polymeric systems, vol. 15. New York: Interscience Publishers,
1962, and M. B. Huglin et. al., "Swelling Properties Of Copolymeric
Hydrogels Prepared By Gamma Irradiation," Journal of Applied
Polymer Science, vol. 31, pp. 457-475, 1986. Immediately following
synthesis, polymeric networks are glassy in the absence of water
and have properties similar to those of other glassy polymers. Upon
water exposure, the crosslinked polymer networks can absorb up to
several times their own weight of water. The hydrophilic nature of
the individual monomers allows absorption of water and the
crosslinked network-like structure of hydrogels prevents easy
release of the absorbed water. This has an impact on the longevity
of the biosensor in that its performance degrades as the biolayer
dries out. It is also important to note that uncrosslinked polymers
synthesized from hydrophilic monomers may simply dissolve in water
and not retain water. The absorbed water improves the plasticity of
the polymer network and provides gel like qualities to the polymer,
resulting in a hydrogel.
[0054] Traditional methods of forming polymer films, such as spin
coating, result in films that are soft, may not be homogeneous in
thickness or properties, and are difficult to form controllably and
uniformly at thicknesses<300 nm. They often display poor
adhesion, require toxic, flammable solvents for spin casting, and
are frequently unstable over time, rendering them unsuitable for
high volume manufacturing. Improvement in stability can be achieved
through an additional fabrication step involving radiation exposure
to crosslink the film, but device sensitivity may be compromised,
as noted by, D. Kuckling, et. al., "Photo-cross-linkable PNIPAAm
copolymers, Synthesis and characterization of constrained
temperature-responsive hydrogel layers," Macromolecules, vol. 35,
pp. 6377-6383, 2002. Alternatively, hydrogel molecules can be
chemically or physically grafted onto surfaces by the use of a
coupling agent followed by solution polymerisation, as outlined by
L. Liang, et. al., "Surfaces with reversible
hydrophilic/hydrophobic characteristics on cross-linked
poly(N-isopropylacrylamide) hydrogels," Langmuir, vol. 16, pp.
8016-8023, 2000. This approach complicates the manufacturing
process and requires the development of specific coupling agents
for different device surfaces, thereby inhibiting efficient
manufacturing.
[0055] Plasma polymerization refers to the formation of films from
plasmas of organic precursors or monomers as outlined by R.
d'Agostino, Plasma deposition, treatment, and etching of polymers,
Boston: Academic Press, 1990, and by H. Yasuda, Plasma
polymerization. Orlando, Fla.: Academic Press, 1985. Energetic
species, for example free radicals, ions, electrons, and excited
state species, are formed in the plasma by electron impact
collisions which cause ionization and fragmentation of the organic
precursors. These species react with other energetic species in the
gas phase and at the surface of a substrate to form a polymer film.
Since plasma-deposited polymers are formed through chemical
reactions at surface sites in a radiation environment, the
resulting films do not resemble conventional polymers which contain
a single repeat unit. Instead, these materials are randomly
crosslinked when deposited, are pinhole free and homogeneous, have
excellent adhesion to substrates or other films and are chemically
and mechanically stable. The films are therefore robust and will
withstand extended storage and operate reliably for long periods of
time. As a result, plasma deposition techniques are attractive
alternatives to spin-casting methods. Recently, acoustic wave
devices have been described by Avramov, I. D., et. al.
Investigations on plasma polymer coated SAW and STW resonators for
chemical gas sensing applications. IEEE Trans. Microwave Theory
Tech. 2001, 49, (4), 827-837, which display high-resolution and
improved detection limits with plasma-deposited polysiloxane,
polystyrene, or polyallyl-alcohol films since they do not
significantly degrade the Q of acoustic devices.
[0056] Preliminary results from investigations by co-inventors Hess
and Hunt have indicated similar improvements for plasma-deposited
hydrogel thin films relative to spin-cast hydrogel thin films in
acoustic wave devices for analyte detection. Two surface acoustic
wave resonator samples were plasma coated with the
N-Isopropylacrylamide (NIPAAm) thin films. The parameters were then
measured using a network analyser and are shown in Table 1. Neither
the insertion loss changes nor phase angle deviations at the center
frequency were dramatically degraded after the film coatings.
TABLE-US-00001 TABLE 1 SAW Device Response before and after
Plasma-deposited hydrogel coating Sample 1 Sample 2 Center Center
freq Insertion Phase freq Insertion Phase (MHz) loss (dB) (deg)
(MHz) loss (dB) (deg) Uncoated 249.60 -7.9 -4.9 249.57 -12.06 0
Coated 248.10 -11.13 -19.7 248.13 -13.53 -8.1 Change -1.5 -3.23
-14.8 -1.44 -1.47 -8.1
[0057] Crosslinked hydrogel thin films can be prepared in a single
step using plasma polymerization, without the use of crosslinkers
or adhesion promoters. FIG. 2 shows prior art methods of
crosslinked hydrogel thin film deposition 200 using (a) the plasma
polymerisation method 202 and (b) the spin coating method 204. For
the plasma polymerisation method 202, a top electrode 210 is
positioned at a predetermined distance from the bottom electrode
211 within a plasma deposition reactor unit (not shown). A suitable
substrate 220 such as, but not limited to, the piezoelectric
material 110 of FIG. 1, is placed in contact with the bottom
electrode 211. An RF voltage supply 215 is electrically connected
to the top electrode 210 and the bottom electrode 211 is
electrically connected to the system ground 216. When certain
conditions within the plasma deposition reactor unit (not shown)
are achieved, such as a specific pressure and temperature, the RF
voltage supply 215 is activated to produce a plasma of organic
monomer and a plurality of electrons, ions and neutrals 225. The
resulting ion bombardment 227 builds up a controlled layer of
hydrogel thin film 228 on the substrate 220. Higher RF voltages 215
and lower pressures within the plasma deposition unit enhance
chemical reactions at the surface of the substrate 220 due to the
resulting higher electron energy and an enhanced ion bombardment
227, and thus promote crosslinking in the deposited hydrogel thin
film 228 as well as improve adhesion to surfaces. These changes
enhance the stability and toughness of the hydrogel thin film 228.
Similarly, co-inventors Hess and Hunt have observed that higher
temperature of the substrate 220 enhance the crosslink densities
and promote better adhesion of the plasma-deposited hydrogel thin
film 228 to the substrate 220. It should be noted that the
substrate 220 is not limited by its geometrical shape or size with
the plasma polymerisation method 202.
[0058] The spin coating method 204 outlined in FIG. 2(b) is a
two-step process. Step 1 places the spin coat precursor 230 on the
substrate 220 which is secured to a spinable plate 231. The
precursor solution is a mixture of monomer and crosslinker 238. The
spinning process spreads the precursor solution 238 over the
substrate 220. Variables such as placement of the precursor
solution 238, geometrical shape and size of the substrate 220,
temperature of the precursor solution 238 and substrate 220 and the
acceleration and speed of the spinable plate 231 are difficult to
control and the final thickness of the precursor solution film 238
is non-uniform and therefore unpredictable and inconsistent. Step 2
involves the separate process of inducing the crosslinking 240. The
precursor solution 238 is exposed to an ultra violet (UV) source
245 for a predetermined amount of time to produce the final
hydrogel thin film 248.
Electrophoretic Technique (Present Invention)
[0059] The present invention is based on a three-dimensional
approach to the immobilization of various molecular recognition
elements instead of the usual two-dimensional surface approach.
Although plasma-deposited polymers can provide a stable surface on
which to immobilize various MREs, the possibility of incorporating
and trapping MREs in the voids of plasma-polymerized hydrogels is
most attractive. With the present invention, MREs can be
incorporated by a novel electrophoretic approach which enables
biomolecules to be either physically trapped or covalently bound to
functional groups within a plasma deposited hydrogel by using
elctrophoretic methods. After drifting the MREs into a hydrogel
thin film using an electrophoretic method, the MREs will be unable
to diffuse out of the film without the application of a sufficient
electric field.
[0060] The electrophoretic technique in accordance with this
invention comprises the controlled manipulation of certain
particles and molecules which contain polar chemical moieties by
the use of an applied electric field. Experiments conducted by a
group of the co-inventors in this application using the
electrophoretic approach have demonstrated that antibodies are
charged sufficiently to permit them to be transported (drifted)
into a plasma-deposited hydrogel under the action of a relatively
low electric field applied to the metal structure of an acoustic
wave device.
[0061] Antibodies are one form of biomolecules with a very complex
three-dimensional structure which recognize a specific antigen
unique to their target. FIG. 3 is a simplified schematic view of an
antibody structure and its fragments 300. Each antibody monomer has
a molecular weight of approximately 150,000 Daltons and is composed
of two identical heavy polypeptide chains 310 and two identical
light chains 320 which are covalently bonded via disulfide (S-S)
linkages between cysteine residues. Each heavy chain 310 is about
440 amino acids long and each light chain 320 is about 220 amino
acids long. An antibody molecule consists of three fragments,
namely two identical Fab fragments 330, which contain antigen
binding sites 332, and one Fc fragment 340 which is the stem of the
antibody and has a carboxyl group 342 on its end. The Fc fragment
region 340 determines the biological properties of the antibody.
Monoclonal antibodies are produced by the descendants of a single B
cell and are identical with each other, as opposed to polyclonal
antibodies, which are derived from multiple cell lines. Monoclonal
antibodies recognize only one specific epitope and have a defined
specificity for the specified antigen. For this reason, monoclonal
antibodies are of more interest for immunosensing biosensor
applications.
[0062] In principle, antibodies, or more generally proteins, can be
fixed noncovalently onto an inert metal surface by simple
adsorption. The inert metal surface would be the metal finger
patterns of interdigital transducers (IDTs) or reflector elements
which constitute the geometries of acoustic wave devices.
Typically, antibodies and other MREs immobilized by physical
adsorption are not stable during the immunosensing process,
especially if buffer rinses are carried out, because the physical
adsorption is based on attraction forces, such as electrostatic
force, rather than chemical bonds. A covalent immobilization is
therefore desired to achieve improved biomolecule activity, reduced
nonspecific adsorption and greater stability. Another key issue
regarding antibody or MRE immobilization is their physical
orientation, which is considered to be a determinant of their
effectiveness. Immobilized antibodies or MREs must be in an
oriented and homogeneous manner, rather than randomly distributed
on the surface of the planar geometries of the sensing device. An
immunoglobulin G (IgG) antibody is considered to be properly
oriented and completely active when immobilized on the Fc fragment
340 of the antibody rather than on the Fab fragments 330, which
contain antigen-binding sites. Therefore, to achieve both stable
and efficient biomolecule binding, it is necessary to couple the
biomolecules to the sensor surface via a heterobifunctional
crosslinker. S. H. Lee "Theoretical and Experimental
Characterization of Time-Dependent Signatures of Acoustic Wave
Based Biosensors", Ph.D Thesis, Georgia Institute of Technology,
2006, has described several antibody and biomolecule immobilizing
methods, such as Alkane-thiol Self Assembled Monolayer (SAM), SPA
(Protein A)+Hydrogel and Offset Lithography Printing (OLP). These
methods are currently employed for the fabrication of small
quantities, but are not practical for high volume manufacturing and
do not produce repeatable biolayers.
[0063] A new approach implementing electrophoretic techniques in
accordance with the invention for the incorporation of antibodies
and other MREs including aptamers into hydrogels will now be
described. This approach is used to incorporate and immobilize
antibodies in a plasma-deposited hydrogel on an acoustic wave
device by immersing the acoustic wave device into a buffer solution
containing antibodies. An electrophoretic driving voltage is then
applied between the SAW bonding pad and an electrode immersed in
the buffer solution. Since the thickness of the hydrogel will be in
the hundreds of nanometer range or less, the driving voltage needed
is low (a few volts) and the transport time short (minutes).
[0064] FIG. 4 is a schematic of electrophoretic immobilization
apparatus for an acoustic wave device 400 in accordance with one
embodiment of the invention. A container 410 contains a portion of
the acoustic wave structure 420 and a buffer solution 450, such
that the buffer solution 450 comes into contact with and completely
envelops the top portion of the hydrogel 430 and the metal
structure 428 which are positioned on the piezoelectric material
424 which forms a portion of the acoustic wave structure 420. The
buffer solution 450 contains the specific antibody molecules 440
required to be deposited onto selected sections of the acoustic
wave structure 420. Other specific molecular recognition elements
may replace the antibody molecules 440 within the buffer solution
450 for other biosensor applications. An electrical stimulus 460 is
placed in close proximity to the container 41 0. The negative
electrode 464 of the electrical stimulus 460 is placed in contact
with the buffer solution 450. The positive electrode 466 of the
electrical stimulus 460 is placed in contact with the metal
structure 428 of the acoustic wave device 420.
[0065] Electrophoretic incorporation of molecular recognition
elements in hydrogel thin films in accordance with the invention is
simple to implement and can be used to control the orientation of
antibodies in the hydrogel. In the electrophoretic immobilization
apparatus 400 shown in FIG. 4, the negatively charged F, portion of
the antibody 340 of FIG. 3 is attracted to the positively biased
metal structure 428 underlying the hydrogel 430, which leaves the
F.sub.ab portion 330 to bind the specific antigen. Furthermore,
since electrophoretic transfer allows incorporation of the antibody
molecules 440 into the three-dimensional network of the hydrogel
430, higher densities of molecular recognition agents can be
achieved using this technique than when the antibodies are
covalently bonded to only the two-dimensional surface of the
hydrogel, as in other antibody and biomolecule immobilizing methods
such as Alkane-thiol Self Assembled Monolayer (SAM), SPA (Protein
A)+Hydrogel and Offset Lithography Printing (OLP). The higher
densities of the molecular recognition centers can be controlled by
adjusting the potential difference of the electrical stimulus 460
and the time duration that the electrical stimulus 460 is
applied.
[0066] A specific example carried out by a group of the inventors
of this application will now be described.
[0067] Fluorescein isothiocyanate antibodies (mouse monoclonal
anti-FITC) were immobilized in the plasma polymerized hydrogel thin
films on acoustic wave resonator devices from a 4% solution of
anti-FITC in 1X-TAE (Tris acetate EDTA) buffer. When diluted in
1X-TAE buffer (pH.about.8.3 at 25.degree. C.), anti-FITC
(pI.about.7.0) takes on a negative charge and hence migrates toward
the positive electrode. To immobilize anti-FITC, the hydrogel
coated acoustic wave resonator device was immersed in the buffer
solution containing anti-FITC and the aluminum bonding pads of the
acoustic wave resonator device were positively biased (0.5-5V DC)
with respect to the buffer solution for 15 min. Subsequent studies
indicated that times less than 5 min are sufficient. Electrical
contact was achieved through micro-DC probes (Alessi) in a probe
station (Cascade MicroTech 9000 Analytical Probe Station). The
experiment was continuously monitored through an attached
microscope (Olympus SZ-60). A fluorescent immunoassay protocol was
then used to confirm the incorporation of anti-FITC into the
hydrogel thin film. Without the presence of an electric field
during exposure to anti-FITC, no antibody incorporation
occurred.
[0068] After the electrophoretic transfer process, several of the
acoustic wave resonator devices were exposed to uranine vapor,
which has a similar chemical structure to FITC, but is liquid at
room temperature, thereby allowing the vapor to be introduced to
the hydrogel/anti-FITC layer by bubbling nitrogen gas through the
uranine container. The hydrogel surface was then washed with the
buffer solution to remove unbound antigen and viewed using a Zeiss
LSM150 confocal fluorescent microscope (CLSM). Application of
voltages between 0.5 and 5 V to the electrode were sufficient to
incorporate the antibody, as indicated by significant differences
in fluorescence intensity from films on the acoustic wave resonator
device. As a result, it was concluded that the fluorescent analyte
is bound to the antibody immobilized in the hydrogel thin film and
therefore that the electrophoretic transfer technique in accordance
with the invention is viable.
[0069] This example has described how an MRE such as fluorescein
isothiocyanate antibodies (anti-FITC) were immobilized in the
plasma polymerized hydrogel thin films using an electrophoretic
method on acoustic wave resonator devices. The basis of this
example can also be used for the forced immobilization of MREs and
other charged molecules onto several other types of films,
including spin-cast or vapor-deposited films. If biomolecules and
other charged molecules diffuse readily into the films without an
extra thrust from an electric field, then the biomolecules and
other charged molecules would just as easily diffuse out of the
film when placed in a solution or other fluid setting, such as from
an implant device into a live body. This invention would also be
suitable for the incorporation of enzymes or other catalysts or
affinity capture agents for application in microchemical
reactors/affinity precipitation or separation devices.
Acoustic Wave Structure Adaptation
[0070] Acoustic wave devices require a metal structure on the free
surface of the piezoelectric material to both generate and detect
the acoustic waves. Details of these metal structures are described
by C. K. Campbell, Surface Acoustic Wave Devices for Mobile and
Wireless Communications, Academic Press, 1998 and by S. Datta,
Surface Acoustic Wave Devices, Prentice-Hall, 1986. These metal
structures also function as a rostrum on which the
antibody-containing hydrogel thin films reside. The metal
structures can easily be adapted to allow for the formation of
localized electrodes which will confine the placement of different
MREs to certain areas within the acoustic wave structure during the
electrophoretic process.
[0071] Acoustic wave biosensors can be operated in an oscillator
configuration, as outlined by S. H. Lee, et al, "Vapor Phase
Detection of Plastic Explosives Using a SAW Resonator Immunosensor
Array," 2005 IEEE Sensors Conference, pp. 468-471, Urvine, Calif.,
2005. The oscillator configuration has an acoustic wave device
which is suitably placed within an electronic circuit such that the
parameters of the acoustic wave device are chosen to meet the
criteria for a positive feedback oscillator relationship to exist.
This relationship, known as the Barkhausen criteria, stipulates the
gain and phase of the oscillator loop. The magnitude and phase
responses of the acoustic wave device will influence the operation
of the oscillator. Several different oscillator configurations can
exist for a variety of acoustic wave devices such as a bulk
acoustic wave (BAW) device, an acoustic wave delay line device and
an acoustic wave resonator type device.
[0072] A simple schematic view of an AT-cut quartz plate bulk
acoustic wave device 500 is shown in FIG. 5. An AT-cut quartz
crystal plate 510 with a diameter of between 13 mm and 14 mm and a
thickness of about 0.17 mm is shown and would normally produce a
fundamental operating frequency of approximately 10 MHz. A front
metal structure 520 usually of gold 100 nm thick is deposited on
the surface of the crystal plate 520. A similar rear metal
structure 530 of the same material and dimensions as the front
metal structure 520 is deposited on the rear of the crystal plate
510. A left electrode 540 connects to the front metal structure 520
and also provides support for the crystal plate 510. Similarly, a
right electrode 550 connected to the rear metal structure 530 and
also provides support for the crystal plate 510. After a suitable
hydrogel layer has been deposited on the front of the crystal plate
510, an electrophoretic driving voltage is then applied to the left
electrode 540 and connects to the front metal structure 520, with
the biomolecule buffer solution serving as the opposite polarity.
This process permits the post-deposition of MREs from biomolecule
buffer solutions into hydrogel thin films in a well-controlled and
reproducible process. Alternatively, after a suitable hydrogel
layer has been deposited on the rear of the crystal plate 510, an
electrophoretic driving voltage is then applied to the right
electrode 550 and connects to the rear metal structure 530 with the
biomolecule buffer solution serving as the opposite polarity. As
binding occurs between the target molecules and the MREs embedded
within the hydrogel layer positioned on the surface of the AT-cut
quartz plate bulk acoustic wave device 500, the acoustic wave will
be perturbed, resulting in a change in its magnitude and phase
response, thereby producing a detectable frequency change within
the oscillator circuit.
[0073] Although the foregoing description related to a specific
type of BAW, such as an AT-cut quartz plate, a similar description
would apply for an SC-cut quartz crystal and other similar BAW
devices.
[0074] It has been shown by S. H. Lee, "Theoretical and
Experimental Characterization of Time-Dependent Signatures of
Acoustic Wave Based Biosensors", Ph.D Thesis, Georgia Institute of
Technology, 2006 that, as the frequency of the oscillator
increases, the sensitivity of the biosensor also increases. The
foremost way to accomplish this is to replace the BAW device with a
surface acoustic wave (SAW) type of device within the oscillator
circuit. One example of a SAW device which would operate as the
feedback element within an oscillator circuit is the acoustic wave
delay line structure 600 shown in FIG. 6. The input interdigital
transducer (EDT) 610 has two sets of adjacent metal strips. The
input DDT pad #1 611 connects to one set of the metal strips of
input IDT 610 and the input IDT pad #2 612 connects to the other
set of metal strips of input IDT 610. As shown, in normal metal
structures for acoustic waves, the pads are in electrical contact
with the metal strips of the IDTs. The metal strips are normally
open circuit with respect to each other. Similarly, the output IDT
620 has a similar metal structure to the input IDT 610. Both the
input IDT 610 and output IDT 620 all reside on the surface of an
appropriate piezoelectric material 630. After a suitable hydrogel
layer is deposited over the input IDT 610 and output IDT 620 and
remaining piezoelectric material 630 therebetween, an
electrophoretic driving voltage is applied either selectively or in
total to the input IDT pad #1 611, input IDT pad #2 612, output IDT
pad #1 621 and output IDT pad #2 622 with the biomolecule buffer
solution serving as the opposite polarity. For the purpose of
electrical connections, the area of the input IDT pad #1 611, input
IDT pad #2 612, output IDT pad #1 621 and output pad #2 622 would
be void of any hydrogel material, thereby exposing the metal for
connection purposes. FIG. 6 depicts IDTs with quarter-wavelength
finger geometries, but this method also works equally well for IDTs
with eighth-wavelength or other suitable finger geometries.
[0075] Another device suitable for an oscillator circuit is the SAW
two-port resonator structure 700 shown in FIG. 7. The input
interdigital transducer (IDT) 710 has two sets of opposite polarity
metal strips. The input IDT pad #1 711 connects to one set of metal
strips and the input IDT pad #2 712 connects to the other set of
metal strips. The metal strips are normally open circuit with
respect to each other. Similarly, the output IDT 720 has a similar
metal structure to the input IDT 710. Adjacent to the input IDT 710
is reflector #1 730, and similarly adjacent to the output IDT 720
is reflector #2 740. As shown, and in normal metal structures for
acoustic waves, the pads are in electrical contact with the metal
strips of the IDTs and reflectors. The input IDT 710, output IDT
720, reflector #1 730 and reflector #2 740 are all located on the
surface of an appropriate piezoelectric material 705. After a
suitable hydrogel layer has been deposited over the input IDT 710,
output IDT 720, reflector #1 730 and reflector #2 740 and remaining
piezoelectric material 705 therebetween, an electrophoretic driving
force is applied either selectively or in total to the input IDT
pad #1 711, input IDT pad #2 712, output IDT pad #1 721, output IDT
pad #2 722, reflector #1 pad 735 and reflector pad #2 745, with the
biomolecule buffer solution serving as the opposite polarity. For
the purpose of electrical connections, the area of the input IDT
pad #1 711, input IDT pad #2 712, output IDT pad #1 721 and output
pad #2 722 is void of any hydrogel material, therefore exposing the
metal for connection purposes. The shape of the magnitude and
frequency response, which in turn controls the operational
frequency of the oscillator circuit, is a function of the reference
distance D 1 751, namely the distance between the input IDT 710 and
output IDT 720, the reference distance D2 752, namely the distance
between the input IDT 710 and reflector #1 730, and the reference
distance D3 753, namely the distance between the output IDT 720 and
reflector #2 740.
[0076] A modified resonator device also suitable for an oscillator
circuit is the SAW two-port resonator structure with an energy
trapping film 800 shown in FIG. 8. The metal structure illustrated
in FIG. 8 with the energy trapping film 800 is more suitable for
use with surface skimming bulk wave (SSBW) type devices as
described by D. L. Lee, "S-BAND SSBW DELAY LINES FOR OSCILLATOR
APPLICATIONS" pp. 245-250, IEEE, Ultrasonics Symposium, 1980. The
input interdigital transducer (IDT) 810 has two sets of opposite
polarity metal strips. The input IDT pad #1 811 is connected to one
set of the metal strips and the input IDT pad #2 812 is connected
to the other set of metal strips. The metal strips are normally
open circuit with respect to each other. The output IDT 820 has a
similar metal structure to the input IDT 810. Reflector#1 830 is
located adjacent to the output IDT 810, and similarly reflector #2
840 is located adjacent to the output IDT 820. As in normal metal
structures for acoustic waves, the pads are in electrical contact
with the metal strips of the IDTs and reflectors and with the
energy trapping film 860. The input IDT 810, output IDT 820,
reflector #1 830 and reflector #2 840 are all located on the
surface of an appropriate piezoelectric material 805.
[0077] After a suitable hydrogel layer has been deposited over the
input IDT 810, output IDT 820, reflector #1 830, reflector #2 840
and energy trapping film 860 and remaining piezoelectric material
805 therebetween, an electrophoretic driving voltage is applied
either selectively or in total to the input IDT pad #1 811, input
IDT pad #2 812, output IDT pad #1 821, output IDT pad #2 822,
reflector #1 pad 835, reflector pad #2 845 and energy trapping film
pad 865, with the biomolecule buffer solution serving as the
opposite polarity. For effecting electrical connections, the input
IDT pad #1 811, input IDT pad #2 812, output IDT pad #1 821 and
output pad #2 822 and energy trapping film pad 865 are void of any
hydrogel material, thereby exposing the metal for connection
purposes. The shape of the magnitude and frequency response, which
in turn controls the operational frequency of the oscillator
circuit, is a function of the width of the energy trapping film 860
the distance between the input IDT 810 and output IDT 820, the
reference distance D2 852, namely the distance between the input
IDT 810 and reflector #1 830, and the reference distance D3 853,
namely the distance between the output IDT 820 and reflector #2
840.
[0078] U.S. Pat. No. 7,053,524 (Edmonson et al.), issued May 30,
2006, the contents of which are hereby incorporated herein by
reference, also describes a surface acoustic wave sensor or
identification device with biosensing capability. This
RFID/biosensor does not rely upon an oscillator circuit for target
detection but responds to an interrogation signal. The
RFID/Biosensor returns a modified interrogation signal resulting
from acoustic wave device parameter changes due to specific binding
events of the biomolecules within the RFID/Biosensor structure.
[0079] A major advantage of an RFID/Biosensor is its ability to
have multiple detecting areas on the same acoustic wave device
which are capable of each having different detection biomolecules.
In the case of an oscillator-based system, the frequency of the
oscillator will change when biomolecule detection occurs, but the
oscillator circuit cannot differentiate between the changes
resulting from having different detection biomolecules on the same
acoustic wave device. One method would be to have several
oscillator circuits, each with a single detection biomolecule and
then combine each of the different frequency changes of the
difference oscillators via an algorithm for multiple analyte
detection, U.S. patent application Ser. No. 11/088809, filed Mar.
25, 2005 by Edmonson et al. and entitled DIFFERENTIATION AND
IDENTIFICATION OF ANALOGOUS CHEMICAL OR BIOLOGICAL SUBSTANCES WITH
BIOSENSORS.
[0080] A step-and-repeat process would entail placing an
electrophoretic voltage on selective areas of the metal structure
of the RFID/biosensor while the structure is immersed in different
biomolecule buffer solutions. This would then position different
MREs on different sections of the metal structure of the
RFID/biosensor. A schematic view of a reflective type
RFID/biosensor with multiple reflector arrays 900 is shown in FIG.
9. An input/output IDT 910 both receives and transmits
electromagnetic signals and transmits incident acoustic waves 920
and receives reflective acoustic waves 925. Normally, input/output
IDT pad #1 915 and input/output pad #2 917 would be interfaced to a
wireless mode via an antenna or a wired mode via a set of suitable
wires. Several suitable reflective arrays are then positioned
within the wave paths of the incident and reflective acoustic waves
920, 925. Although FIG. 9 shows the reflective arrays on one side
of the input/output IDT 910, an alternative structure may have
reflector arrays positioned on either or both sides of the
input/output IDT 910 due to the bi-directional nature of the
incident acoustic wave 920. FIG. 9 shows IDTs with
quarter-wavelength finger geometries but IDTs with
eighth-wavelength or other suitable finger geometries may be used.
Reflector array A 930 and its reflector array A pad 935, reflector
array B 940 and reflector array B pad 945 and reflector array "n"
950 and its reflector array "n" pad 955 are suitably positioned
with respect to the input/output IDT 910 and the incident acoustic
waves 920. As in normal metal structures for acoustic waves, the
pads are in electrical contact with the metal strips of the IDTs
and reflectors.
[0081] The input/output IDT 910, reflector array A 930, reflector
array B 940 and up to and including reflector array "n" 950 are all
located on the surface of an appropriate piezoelectric material
905. After a suitable hydrogel layer has been deposited over the
input/output IDT 910, reflector array A 930, reflector array B 940
and up to and including reflector array "n" 950 and remaining
piezoelectric material 905 therebetween, an electrophoretic driving
voltage is applied either selectively or in total to the
input/output IDT pad #1 915, input/output IDT pad #2 917, reflector
array A pad 935, reflector array pad B 945 and up to and including
reflector array pad "n" 955 with the same or different biomolecule
buffer solutions serving as the opposite polarity. For the purpose
of effecting electrical connections, the input/output IDT pad #1
915, input/output IDT pad #2 917, reflector array A pad 935,
reflector array pad B 945 and up to and including reflector array
pad "n" 955 are void of any hydrogel material, thereby exposing the
metal for connection purposes. By placing different MREs on the
various different areas of reflector array A 930, reflector array B
940 and up to and including reflector array "n" 950, a selective
detection and identification algorithm within the system receiving
the modified interrogation signal can discern the different binding
events at the different MRE sites.
[0082] Another metal structure described in U.S. Pat. No. 7,053,524
which is used for RFID/Biosensors is one which utilizes an IDT
array instead of a reflector array. FIG. 10 shows the basic
elements and structure of an RFID/biosensor with selectable IDT
arrays 1000. An input/output IDT 1010 both receives and transmits
electromagnetic signals and transmits and receives acoustic waves
1012. In use, input/output IDT pad #1 1015 and input/output pad #2
1017 are interfaced to a wireless mode via an antenna or a wired
mode via a set of suitable wires. Several suitable IDT arrays
sections are then suitably positioned within the acoustic wave
path. Although FIG. 10 shows the IDT array sections on one side of
the input/output IDT 1010, an alternative structure may have IDT
array sections positioned on either or both sides of the
input/output IDT 1010 due to the bi-directional nature of the
acoustic waves 1012. FIG. 10 shows IDTs with quarter-wavelength
finger geometries but IDTs with eighth-wavelength or other suitable
finger geometries may be used. IDT array section #1 1020 and IDT
array section pad A 1022 and IDT array section pad B 1024, IDT
array section #2 1030 and IDT array section pad C 1032 and IDT
array section pad D 1034, IDT array section #3 1040 and IDT array
section pad E 1042 and IDT array section pad F 1044 and up to and
including IDT array section #n 1050 along with its IDT array
section pad n, 1052 and IDT array section pad ny 1054, are suitably
positioned with respect to the input/output IDT 1010 and acoustic
waves 1012. As in normal metal structures for acoustic waves, the
pads are in electrical contact with the metal strips of the
IDTs.
[0083] The input/output IDT 1010, IDT array section #1 1020, IDT
array section #2 1030, IDT array section #3 1040 and up to and
including IDT array section "n" 1050 are all located on the surface
of an appropriate piezoelectric material 1005. After a suitable
hydrogel layer has been deposited over the input/output IDT 1010,
IDT array section #1 1020, IDT array section #2 1030, IDT array
section #3 1040 and up to and including IDT array section "n" 1050
and remaining piezoelectric material 1005 therebetween, an
electrophoretic driving voltage is then applied either selectively
or in total to IDT array section pad A 1022 and IDT array section
pad B 1024, IDT array section pad C 1032 and IDT array section pad
D 1034, IDT array section pad E 1042 and IDT array section pad F
1044 and up to and including IDT array section pad n, 1052 and IDT
array section pad n.sub.y 1054 with the same or different
biomolecule buffer solutions serving as the opposite polarity.
[0084] For the effecting electrical connections, the input/output
IDT pad #1 1015, input/output IDT pad #2 1017, IDT array section
pad A 1022 and IDT array section pad B 1024, IDT array section pad
C 1032)and IDT array section pad D 1034, IDT array section pad E
1042 and IDT array section pad F 1044)and up to and including IDT
array section pad n.sub.x 1052 and IDT array section pad n.sub.y
1054 are void of any hydrogel material, thereby exposing the metal
for connection purposes. By placing different MREs on the various
different areas of IDT array section #1 1020, IDT array section #2
1030, IDT array section #3 1040 and up to and including IDT array
section "n" 1050, a selective detection and identification
algorithm within the system receiving the modified interrogation
signal can discern the different binding events at the different
MRE sites.
[0085] The operation of certain types of RFID/Biosensors as
described in U.S. Pat. No. 7,053,524 require that, for some
particular types, all of the IDTs be connected to common
connectivity points. FIG. 11 is a schematic outlining the inter-IDT
pad connectivity 1100. The acoustic wave device is attached to a
printed circuit material base 1110 together with a suitable metal
circuit trace A 1120 and a metal circuit trace B 1125. The acoustic
wave device is positioned on or within the printed circuit material
base 1110 in a manner such that the pads of the acoustic wave
device, namely pad #1 1141, pad #2 1142, pad #3 1143, pad #4 1144
and up to and including pad X 1145 and pad Y 1146, located on the
piezoelectric material 1130 are in close proximity to the metal
circuit trace A 1120 and the metal circuit trace B 1125. Metal
interconnects 1150 complete an electrical circuit between pad #1
1141, pad #2 1142, pad #3 1143, pad #4 1144 and up to and including
pad X 1145 and pad Y 1146 and the metal circuit traces A 1120 and B
1125. This metal interconnects may be solder, bond wires,
conductive epoxy or other small conductive circuit components.
[0086] One problem which multiple reflector or IDT arrays such as
described in U.S. Pat. No. 7,053,524 encounter, when a method such
as described with reference to FIG. 4 is used, is the contamination
of different molecular recognition elements on adjacent reflector
or IDT arrays of the RFID/Biosensor. If for example molecular
recognition element A is required to be placed on IDT array A and
molecular recognition element B is required to be placed on IDT
array B using a two step procedure and similar apparatus to that
shown in FIG. 4, there is a high probability that some residue of
molecular recognition element A will be on IDT B and
vice-versa.
[0087] A method to avoid cross-contaminating the molecular
recognition elements on different reflector or IDT arrays using a
multi-electrode electrophoretic immobilization assembly 1200 is
shown in FIG. 12. A container 1210 contains the acoustic wave
device 1220 and a buffer solution 1250 such that the buffer
solution 1250 contacts and completely bounds the top portion of the
hydrogel 1230 and an array #1 1222 and an array #2 1226 positioned
on the piezoelectric material 1224 which forms the acoustic wave
structure. The buffer solution 1250 contains the specific molecular
recognition elements (MREs) 1240 required to be deposited into the
hydrogel 1230, which is located in the close proximity of array #2
1226 of the acoustic wave structure. An electrical stimulus 1260 is
conveniently placed close to the container 1210. A negative
electrode #1 1262 of the electrical stimulus 1260 is placed in
contact with array #1, and a negative electrode #2 1264 of the
electrical stimulus 1260 is placed in contact with the buffer
solution 1250. The positive electrode 1266 of the electrical
stimulus 1260 is placed in contact with array #2 1226. The
molecular recognition elements will be attracted to the hydrogel
1230 located near the more positive metal structure of array #2
1226 and repelled away from the more negative metal structure of
array #1 1222.
[0088] This embodiment shows how the multi-electrode
electrophoretic immobilization assembly 1200 functions for two
metal structure arrays and can easily be expanded to accommodate a
multiplicity of metal structure arrays by providing electrical
contact by the multiple metal structure arrays with a set of
multiple electrodes, all of which are connected to the most
negative electrode 1262.
General Electrophoretic Technique
[0089] Many other types of devices utilize a thin film embedded
with biomolecules for a variety of applications. The described
method of preparing thin films using an electrophoretic technique
can be expanded to include other devices which require their
biomolecules not to diffuse out of the film when placed in a
solution. The electrophoretic technique in accordance with the
present invention forces the biomolecules into the film by use of
an electric field and thereby accomplishes the correct orientation
of the biomolecules and prevents them from diffusing away from the
film in the absence of any electric field.
[0090] Certain thin film materials can be re-engineered by the
introduction of biomolecules either onto their surfaces or embedded
within the thin film material itself. Such thin film materials have
made their way into applications such as biosensors, drug delivery
systems, flexible electronics and protective coatings, to name a
few.
[0091] A optical biosensor described by Sandstrom, T., Steinberg,
M. & Nygren, H. (1985) Appl. Opt. 24,472-479, is capable of
transforming certain molecular interactions into optical signals
due to the additional mass deposited on the thin film surface by
enzymatic catalysis, thereby altering the wavelength of light
reflected by the optical layer. Another type of biosensor which
utilizes a micromechanical design has receptor biomolecules affixed
to the surface of a thin film material which is positioned on a
cantilever. As certain substances are captured by the receptor
biomolecules, the cantilever increases in mass and changes its
vibration frequency.
[0092] Other uses of re-engineered thin films now include drug
delivery schemes, as reported by the Institute for
NanoBioTechnology at Johns Hopkins University, where biomolecules
and nanoparticles are released in a controlled manner by applying a
brief electric field. This invention can also be applied to the
modification of various surfaces for medical implants with
protective biomolecule coatings which are designed to enhance
biocompatibility with surrounding tissue.
[0093] Flexible electronics can make use of thin films which are
embedded with functional biomolecules such as ferritin. These very
small-scale circuits will play an important role in biomolecular
electronics for information processing applications. Another use of
modified thin films is to modify the mechanical and physical
properties of the surface for MEMS and NEMS scale devices. Such
applications focus on providing flexible electronic circuitry
platforms and the wetting, lubrication and protection properties of
the thin films.
[0094] A general thin film structure 1300 receptive to the
electrophoretic technique in accordance with the invention is shown
in FIG. 13. A base material 1310 such as metallic, semiconductor or
other conductive materials, polymers, crystals, ceramics and other
such materials including nanocomposites, can function as the
platform for various devices which require the re-engineering of
their surfaces. A metal structure 1320 is positioned on the base
material 1310 in such a manner that an external electrical probe
can come into contact therewith. A thin film 1330 is deposited over
the metal structure and other remaining areas of the base material
1310 if the application or manufacturing process permits. The
biomolecule material 1340 is then forced into the thin film 1330
using the electrophoretic technique in accordance with the
invention.
[0095] Although the example of a general thin film structure
receptive to the electrophoretic technique 1300 shown in FIG. 13
has only one metal structure 1320, it is a natural extension to
replicate several metal structures to permit different biomolecule
materials to be electrophoretically applied to the respective
structures.
[0096] If for example biomolecule A is required to be placed on
array A and biomolecule B is required to be placed on array B using
a two step procedure and similar apparatus to that shown in FIG. 4,
then there is a high probability that some residue of biomolecule A
will be on array B and vice-versa. A method to avoid
cross-contaminating the different biomolecules on different arrays
using a multi-electrode electrophoretic immobilization assembly
1400 is illustrated in FIG. 14. A container 1410 contains the base
material 1420 and a buffer solution 1450 such that the buffer
solution 1450 contacts and completely bounds the top portion of the
thin film 1430 and a conducting array #1 1422 and a conducting
array #2 1426 positioned on the base material 1420. The buffer
solution 1450 contains the specific biomolecules 1440 required to
be deposited within the thin film 1430 and located in close
proximity to conducting array #2 1426 of the general thin film
structure 1300.
[0097] An electrical stimulus 1460 is conveniently placed close to
the container 1410. The negative electrode #1 1462 of the
electrical stimulus 1460 is placed in contact with conducting array
#1 and the negative electrode #2 1464 of the electrical stimulus
1460 is placed in contact with the buffer solution 1450. The
positive electrode 1466 of the electrical stimulus 1460 is placed
in contact with conducting array #2 1426. The biomolecules will be
attracted to the more positive conducting array #2 1426 and be
repelled away from the more negative conducting array#11422.
[0098] This example shows how the multi-electrode electrophoretic
immobilization assembly 1400 functions for two metal structure
arrays and can easily be expanded to accommodate a multiplicity of
metal structure arrays by effecting electrical contact of the
multiple metal structure arrays with a set of multiple electrodes,
all of which are connected to the most negative electrode 1462.
[0099] The advantages of the invention will now be readily apparent
to a person skilled in the art from the foregoing description of
preferred embodiments. Other advantages and embodiments of the
invention will also now be readily apparent to a person skilled in
the art, the scope of the invention being defined in the appended
claims.
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