Patent Application
20080142366
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.
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.
It is an object of this invention to provide an improved method for incorporating biomolecules in thin films.
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.
The biomolecules may comprise molecular recognition elements (MREs), and the molecular recognition elements may comprise antibodies.
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.
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.
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.
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.
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.
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:
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”.
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”.
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”.
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.
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.
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.
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.
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.
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 Fc portion of an antibody is attracted to a positively biased metal structure underlying a hydrogel film derived from N-Isopropylacrylamide (NIPAAm), which leaves the Fab 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.
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:
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.
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.
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.
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.
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.
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.
K. Länge et al, “A Surface Acoustic Wave Biosensor Concept with Low Flow Cell Volumes for Label-Free Detection,” Anal. Chem., 75 (20), 5561-5566, 2003.
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.
F. Bender et al, “Love-wave biosensors using cross-linked polymer waveguides on LiTaO substrates,” Electronics Letters, Vol. 36, No. 19, 2000.
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.
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.
Embodiments of the invention will now be described, by way of example, with reference to the accompanying drawings, of which:
b),
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.
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, TP 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.
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.
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 (γ-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.
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.
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.
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.
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.
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.
Crosslinked hydrogel thin films can be prepared in a single step using plasma polymerization, without the use of crosslinkers or adhesion promoters.
The spin coating method 204 outlined in
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.
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.
Antibodies are one form of biomolecules with a very complex three-dimensional structure which recognize a specific antigen unique to their target.
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.
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).
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
A specific example carried out by a group of the inventors of this application will now be described.
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˜8.3 at 25° C.), anti-FITC (pI˜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.
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.
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 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.
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.
A simple schematic view of an AT-cut quartz plate bulk acoustic wave device 500 is shown in
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.
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
Another device suitable for an oscillator circuit is the SAW two-port resonator structure 700 shown in
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
After a suitable hydrogel layer has been deposited over the input IDT 810, output IDT 820, reflector #1830, reflector #2840 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 #1811, input IDT pad #2812, output IDT pad #1821, output IDT pad #2822, reflector #1 pad 835, reflector pad #2845 and energy trapping film pad 865, with the biomolecule buffer solution serving as the opposite polarity. For effecting electrical connections, the input IDT pad #1811, input IDT pad #2812, output IDT pad #1821 and output pad #2822 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 D2852, namely the distance between the input IDT 810 and reflector #1830, and the reference distance D3853, namely the distance between the output IDT 820 and reflector #2840.
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.
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.
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
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 #1915, input/output IDT pad #2917, 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 #1915, input/output IDT pad #2917, 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.
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.
The input/output IDT 1010, IDT array section #11020, IDT array section #21030, IDT array section #31040 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 #11020, IDT array section #21030, IDT array section #31040 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 ny 1054 with the same or different biomolecule buffer solutions serving as the opposite polarity.
For the effecting electrical connections, the input/output IDT pad #11015, input/output IDT pad #21017, 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 nx 1052 and IDT array section pad ny 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 #11020, IDT array section #21030, IDT array section #31040 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.
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.
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
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
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.
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.
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.
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.
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.
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.
A general thin film structure 1300 receptive to the electrophoretic technique in accordance with the invention is shown in
Although the example of a general thin film structure receptive to the electrophoretic technique 1300 shown in
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
An electrical stimulus 1460 is conveniently placed close to the container 1410. The negative electrode #11462 of the electrical stimulus 1460 is placed in contact with conducting array #1 and the negative electrode #21464 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 #21426. The biomolecules will be attracted to the more positive conducting array #21426 and be repelled away from the more negative conducting array#11422.
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.
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.
