Patent Publication Number: US-9417216-B2

Title: Atomic layer deposition inverted passivated surface acoustic wave sensor for early detection of biofilm growth

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of, and priority to, U.S. Provisional Patent Application Ser. No. 61/753,332, filed on Jan. 16, 2013, entitled “ALD Passivated Bacterial Biofilm Sensor Using Inverted Surface. Acoustic Wave” by Young Wook Kim et al., the entire contents of which is hereby incorporated by reference herein. 
    
    
     STATEMENT OF GOVERNMENT SUPPORT 
     This invention was made with U.S. government support under EFRI1042881 awarded by the National Science Foundation. The U.S. government has certain rights in the invention. 
    
    
     BACKGROUND 
     1. Technical Field 
     The present disclosure relates to surface acoustic wave (SAW) sensors for detecting the growth of biofilms. More particularly, the present disclosure relates to piezoelectric SAW sensors having passivation film layer. 
     2. Background of Related Art 
     Bacteria can attach to surfaces and form microcolonies as their population increases. The colonies eventually can form a community known as a bacterial biofilm [1, 2]. A biofilm is not simply a group of bacteria, but a complex collection of microorganisms encased in an extracellular matrix. The extracellular matrix is composed of exopolysaccharide polymers which promote irreversible adhesion of microcolonies on the surface and also prevent diffusion of antibiotics through the biofilm [1, 2]. Due to the complex extracellular matrix and heterogeneous bacterial composition, biofilms are resistant to bacteriophages in industry and to chemically diverse antibiotic treatments in clinical fields [3]. In addition, bacterial corrosion of metals is an economically important consequence of bacterial biofilm formation that illustrates several fascinating aspects of the structure and physiology of these adherent bacterial populations. Therefore, environmental, clinical, and industrial long term reliable biofilm growth monitoring is critical to prevent contamination, severe infection, and corrosive problems due to the biofilm formation. 
     The measurement of bacterial biofilms with capacitive sensing has been applied by Yang and Li to monitor  Salmonella typhimurium  bacteria [4], and by Ghafar-Zadeh et al. to detect  Escherichia coil  ( E. coli ) [5]. In Yang and Li at al. [4], an interdigitated microelectrode was fabricated to provide detectable impedance signals in capacitance measurement during bacterial growth,  S. typhimurium  bacteria were grown over the microelectrode and the capacitance change was continuously measured. Capacitive sensing in a liquid environment, however, can be interfered with by a conductive media due to the current flow through the growth media [4]. 
     The direct impedance measurement of the attachment of  E. coli  on an electrode is demonstrated by other groups [6, 7]. The change of impedance during bacterial growth is correlated with the biomass adhere on the electrode. This impedimetric sensing is particularly useful in detecting very early attachment of bacteria based on the significant impedance change observed upon attachment. However, long-term real-time biofilm monitoring by impedance measurement requires a continuous current source for bacterial detection which may cause interruption of bacterial growth. 
     Fluorescent methods have reported high sensitivity [8], but require fluorescent molecule labeling for sensing to occur. Labeling requires additional sample preparation and the fluorescent molecule can be degraded over long term exposure to liquid. 
     Electrochemical sensing can be used for selective detection a molecule without fluorescent labeling [9]. An electrochemical sensor array was integrated with a miniaturized bioreactor system for high throughput cell cultivation in 96 well plates [10]. Using a 100 μl working volume in the 96 well micro reactors, the sensor array can monitor temperature, pH, and oxygen concentration as well as total biomass. However, electrochemical sensors require a continuous power source for the operation and also require recalibration of the sensor due to the conductivity change of bacterial growth media in long term biofilm growth experiments. 
     Surface Acoustic Wave (SAW) sensors exhibit several advantages in small molecule detection including high sensitivity [11-22] and low power consumption [23]. A SAW sensor can detect mass or viscosity change due to the wave velocity attenuation, resulting in a resonant frequency shift at the output. A highly sensitive SAW sensor for detection of interleukin-6 (IL-6), which is one of key molecules in human immune system, was reported. In Krishnamoorthy et al. [19], a specific receptor for IL-6 was immobilized on the surface of the SAW sensor. Based on the resonant frequency shift due to the IL-6 binding, the detection limit of the SAW sensor was approximately 10 −18  g (grams). A SAW sensor is also a passive device. 
     The power for operation of the SAW sensor can be delivered by an external device wirelessly which makes the SAW sensor useful for long term biofilm monitoring without a continuous power supply [23]. Furthermore, the SAW sensor can be fabricated using biocompatible materials [24-26]. The combination of extremely high sensitivity, biocompatibility, and low power consumption makes the SAW sensor a unique tool for real time monitoring of bacterial biofilm growth. However, it is also noted that piezoelectric materials used in the SAW sensor can be dissolved due to long term exposure to liquid [27]. 
     SUMMARY 
     The present disclosure relates to a novel inverted passivated SAW sensor for real time biofilm monitoring. A piezoelectric film is deposited by pulsed laser deposition, and the sensor is effectively passivated by a passivated film layer using atomic layer deposition to prevent damage to the piezoelectric layer in bacterial growth media and animal serum. 
     The SAW sensor can be reused after oxygen plasma cleaning, allowing for consecutive biofilm formation experiments using one sensor. 
     Therefore, the present disclosure relates to a novel surface acoustic wave (SAW) biofilm sensor comprising: a SAW transducer; a piezoelectric film layer; and a passivation film layer. The piezoelectric film layer is mounted over the SAW transducer and the passivation film layer is mounted over the piezoelectric film layer. In one embodiment, the passivation layer includes aluminum oxide, Al 2 O 3 . In yet another embodiment, the passivation layer defines a thickness of at least 45 nanometers (nm). In a still further embodiment, the piezoelectric layer includes zinc oxide, ZnO. In yet another embodiment, the piezoelectric layer defines a thickness of at least 40 nanometers (nm). 
     The present disclosure relates also to a surface acoustic wave (SAW) biofilm sensor that includes a transmitting electric to acoustic wave transducer defining an upper surface and a lower surface, a receiving acoustic wave to electric transducer defining an upper surface and a lower surface, a piezoelectric film layer defining an upper surface and a lower surface and a passivation film layer defining an upper surface and a lower surface. A portion of the lower surface of the piezoelectric film layer is disposed on the upper surface of the transmitting electric to acoustic wave transducer and another portion of the lower surface of the piezoelectric film layer is disposed on the upper surface of the receiving acoustic wave to electric transducer and the lower surface of the passivation film layer is disposed on the upper surface of the piezoelectric film layer. The upper surface of the passivation film layer thereby configured to enable contact with a biofilm. 
     In one embodiment, the SAW biofilm sensor may further include a piezoelectric SAW loss reduction film layer defining an upper surface and a lower surface wherein the lower surface of the transmitting electric to acoustic wave transducer is disposed on a portion of the upper surface of the piezoelectric SAW loss reduction film layer and wherein the lower surface of the receiving acoustic wave to electric transducer is disposed on another portion of the upper surface of the piezoelectric SAW loss reduction film layer. 
     In still another embodiment, the SAW biofilm sensor may further include a substrate defining an upper surface and a lower surface, wherein the lower surface of the piezoelectric SAW loss reduction film layer is disposed on the upper surface of the substrate. 
     In yet another embodiment, a portion of the lower surface of the piezoelectric film layer is disposed on a portion of the upper surface of the piezoelectric SAW loss reduction film layer and disposed between the transmitting electric to acoustic wave transducer and the receiving acoustic wave to electric transducer. 
     In a still further embodiment, another portion of the lower surface of the piezoelectric film layer may be disposed between the transmitting electric to acoustic wave transducer and the receiving acoustic wave to electric transducer. 
     In one embodiment of the SAW biofilm sensor, wherein the piezoelectric layer defines an upper sub-layer and a lower sub-layer, wherein the lower sub-layer defined by the portion of the lower surface of the piezoelectric film layer is disposed on the portion of the upper surface of the lower piezoelectric film layer and is disposed between the transmitting electric to acoustic wave transducer and the receiving acoustic wave to electric transducer. The upper sub-layer is defined by the portion of the piezoelectric layer between the passivation film layer and the lower sub-layer. The lower sub-layer has a shear modulus and density to define a first SAW velocity. The upper sub-layer has a shear modulus and density to define a second SAW velocity wherein the second velocity differs from the first velocity. 
     In one embodiment, the second velocity is equal to or greater than the first velocity. 
     In still another embodiment, the SAW biofilm sensor the passivation layer includes aluminum oxide, Al 2 O 3 . In one embodiment, the passivation layer defines a thickness between the upper surface of the passivation layer and the lower surface of the passivation layer wherein the thickness of the passivation layer has a dimension of at least 45 nanometers (nm). 
     In yet another embodiment, the piezoelectric layer may include zinc oxide, ZnO. In one embodiment, the piezoelectric layer defines a thickness between the upper surface of the piezoelectric layer and the lower surface of the piezoelectric layer and the thickness of the piezoelectric layer has a dimension of at least 40 nanometers (nm). 
     The present disclosure relates also to a method of assembling a biofilm surface acoustic wave (SAW) sensor that includes depositing a piezoelectric layer on a SAW transducer electrode pattern and depositing a passivation layer on the piezoelectric layer. 
     In one embodiment, the depositing of the piezoelectric layer may include depositing a layer of zinc oxide ZnO on the SAW transducer electrode pattern. In a still further embodiment, the depositing a layer of zinc oxide ZnO on the SAW transducer electrode pattern may include depositing a layer of zinc oxide ZnO having a thickness of at least 40 nanometers (nm). 
     In yet another embodiment, the depositing a passivation layer on the piezoelectric layer may include depositing a layer of aluminum oxide Al 2 O 3  on the piezoelectric layer. 
     In a still further embodiment, the depositing of the layer of aluminum oxide Al 2 O 3  on the piezoelectric layer may include depositing a layer of aluminum oxide Al 2 O 3  having a thickness of at least 45 nanometers (nm) on the piezoelectric layer. 
     In one embodiment, the method of assembling may further include depositing the SAW transducer electrode pattern on a piezoelectric SAW loss reduction film layer. The method may further include depositing the piezoelectric SAW loss reduction film layer on a substrate. 
     In another embodiment, the step of depositing the piezoelectric layer on the SAW transducer electrode pattern includes pulsed laser deposition. 
     In still another embodiment, the step of depositing the passivation layer on the piezoelectric layer includes atomic layer deposition. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other advantages will become more apparent from the following detailed description of the various embodiments of the present disclosure with reference to the drawings wherein: 
         FIG. 1  illustrates a cross-sectional view of an example of a coated piezoelectric SAW transducer according to the prior art: 
         FIG. 2  illustrates a top perspective view of an exemplary embodiment of an uncoated piezoelectric SAW transducer within a piezoelectric substrate that is utilized to receive a passivation film according to one embodiment of the present disclosure; 
         FIG. 3  illustrates a cross-sectional view of a coated piezoelectric SAW transducer biofilm sensor according to one embodiment of the present disclosure: 
         FIG. 4A  illustrates a top perspective schematic view of an exemplary embodiment of the SAW biofilm sensor of  FIGS. 2 and 3  according to one embodiment of the present disclosure that includes the piezoelectric SAW transducer of  FIG. 2  further including a piezoelectric layer and a passivation layer disposed on the upper surface of the SAW transducer and further illustrating a SAW passing through a biofilm on the upper surface of the passivation layer; 
         FIG. 4B  illustrates a cross-sectional schematic view of the SAW biofilm sensor of  FIG. 4A  illustrating the piezoelectric transducer and the thickness dimensions of the piezoelectric layer, the passivation layer and the biofilm; 
         FIG. 5  illustrates a schematic diagram of pulsed layer deposition of the piezoelectric layer on a substrate for assembling the piezoelectric SAW biofilm sensor of  FIGS. 2, 3, 4A and 4B ; 
         FIG. 6  illustrates a schematic diagram of atomic layer deposition of the passivation layer of  FIGS. 3, 4A and 4B ; 
         FIG. 7A  is a schematic illustration of a piezoelectric SAW loss reduction film layer deposited on a substrate; 
         FIG. 7B  is a schematic diagram of an interdigitated piezoelectric SAW transducer electrode pattern deposited on the piezoelectric SAW loss reduction film layer of  FIG. 7A ; 
         FIG. 7C  is a schematic diagram of a piezoelectric layer deposited on the electrode pattern and the piezoelectric SAW loss reduction film layer deposited on a substrate of  FIGS. 7A and 7B ; 
         FIG. 7D  is a schematic diagram of the piezoelectric layer and electrode pattern of  FIG. 7C  undergoing annealing; 
         FIG. 7E  is a schematic diagram of a passivation layer deposited partially on the electrode pattern of  FIGS. 7B, 7C and 7D  and on the piezoelectric layer of  FIGS. 7C and 7D ; 
         FIG. 8  is a graphical plot of normalized sensitivity of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  for various passivation layer materials including without passivation; 
         FIG. 9  is an optical microscopy image of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 20 nanometers (nm) showing damaged areas; 
         FIG. 10  is an optical microscopy image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 30 nm showing damaged areas; 
         FIG. 11  is an optical microscopy image of yet another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 40 nm showing damaged areas; 
         FIG. 12  is an optical microscopy image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 45 nm showing no damage; 
         FIG. 13  is an optical surface image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by a 45 nm Al 2 O 3  film using e-beam evaporation showing damage; 
         FIG. 14  is an optical surface image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by a 45 nm Al 2 O 3  film using RF sputtering showing damage; 
         FIG. 15  is an optical surface image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by a 45 nm Al 2 O 3  film using ALD showing no damage; 
         FIG. 16  is a top perspective view of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  as a packaged unit together with other components for biofilm sensing according to one embodiment of the present disclosure; 
         FIG. 17  is an optical microscopy image of the surface of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  before biofilm cleaning; 
         FIG. 18  is an optical microscopy image of the surface of the SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  after oxygen plasma biofilm cleaning; 
         FIG. 19  is a graphical plot of negative resonant frequency shift results for SAW biofilm sensors assembled in accordance with  FIGS. 2-7E  versus time due to lysogeny broth (LB) biofilm growth for three newly fabricated SAW transducers; 
         FIG. 20  is a graphical plot of negative resonant frequency shift results for a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  versus time due to lysogeny broth biofilm growth in three sequential growth experiments using one device in an LB; 
         FIG. 21  is a graphical plot of negative resonant frequency shift results for a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  versus time in three growth experiments using a newly fabricated sensor in 10% fetal bovine serum (FBS); 
         FIG. 22  is a graphical plot of averaged biofilm thickness and standard deviation in LB media and in 10% FBS over 30-40 locations measured by optical microscopy; 
         FIG. 23  is a microscopy image of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  after biofilm growth experiments in LB media; 
         FIG. 24  is a microscopy image of the SAW biofilm sensor of  FIG. 23  assembled in accordance with  FIGS. 2-7E  after biofilm growth experiments in 10% FBS; 
         FIG. 25  is a microscopy image of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  after cleaning with deionized water; 
         FIG. 26  is a microscopy image of the SAW biofilm sensor of RIG, 25 after ultrasonication cleaning in acetone for 3 hours; 
         FIG. 27  is a microscopy image of the SAW biofilm sensor of  FIGS. 25 and 26  after oxygen plasma cleaning for 30 seconds; 
         FIG. 28  is a microscopy image of the SAW biofilm sensor of  FIGS. 25, 26 and 27  after a second biofilm growth experiment and oxygen plasma cleaning for 30 seconds; and 
         FIG. 29  is a perspective view a top perspective view of an exemplary embodiment of a SAW biofilm sensor that includes an antenna for radiofrequency (RF) coupling to a radiofrequency identification (RFID) reader. 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure relates to the design of a passivated piezoelectric SAW transducer biofilm sensor according to embodiments of the present disclosure without loss of sensitivity for biosensing applications. 
     First, to illustrate the advantages of the embodiments of the present disclosure,  FIG. 1  illustrates an example of a prior art SAW biofilm sensor  10  wherein the piezoelectric materials used in the SAW biofilm sensor can be dissolved due to long term exposure to liquid. More particularly, SAW biofilm sensor  10  includes a SAW transducer  20  having a transmitting electrode transducer  22  and a receiving electrode transducer  24  each mounted over a common piezoelectric loss reduction film  30  and a substrate  32 . The common piezoelectric loss reduction film  30  and substrate  32  form a transducer mounting structure  35 . The transmitting electrode transducer  22  includes an elevated portion  23  and the receiving electrode transducer  24  includes a corresponding interfacing elevated portion  25  which are each raised above upper surface  30   a  of the common piezoelectric loss reduction film  30 . Both the elevated portion  23  and the elevated portion  25  are elevated above, and not in contact with, the upper surface  30   a  to provide a space above the common piezoelectric loss reduction film  30  for a thin film  26  in contact with, and mounted over, the common piezoelectric loss reduction film  30 . The elevated portions  23  and  25  each extend partially over upper surface  26   a  of the thin film  26 . During usage, a biofilm  40  is positioned over both the elevated portions  23  and  25  and upper surface  26   a  of the thin film  26 . 
     The substrate  32  may be made from silicon Si and the common piezoelectric loss reduction film  30  may be made from silicon dioxide SiO 2 . 
     Since the biofilm  40  is in direct contact with the elevated portions  23  and  25  of the transmitting electrode transducer  22  and the receiving electrode transducer  24 , respectively, the configuration of a SAW sensor such as SAW sensor  10  in  FIG. 1  has been shown to be susceptible to dissolution of the common piezoelectric film  30  due to long term exposure to the liquid biofilm. 
     In contrast, the present disclosure relates to a successfully passivated ZnO based SAW sensor for long term biofilm growth monitoring in an animal serum or bacterial growth media. Atomic Layer Deposition (ALD) was applied for high density and conformal aluminum oxide (Al 2 O 3 ) film deposition to protect the ZnO of the SAW sensor from media. The SAW sensor was used for the in vitro real time study of  E. coli  static biofilm growth in Lysogeny Broth (LB) media and in 10% Fetal Bovine Serum (FBS), the latter of which is the most widely used serum for mammalian cell culture due to similarities to in vivo environments [28]. 
     The remainder of the present disclosure presents the design of the inverted SAW sensor for the targeted mode of the wave and fabrication process. The material and experimental procedures for biofilm detection in the sensor are presented. The results show the biofilm detection using the sensor in consecutive testing both in bacterial growth media and animal serum. 
     More particularly,  FIG. 2  illustrates a top perspective view of an exemplary embodiment of an uncoated piezoelectric SAW transducer  100  having a transmitting electric to acoustic wave transducer  110  and a receiving acoustic wave to electric transducer  110 ′ both mounted on upper surface  30   a  of the transducer mounting substrate  35  of  FIG. 1  that is utilized to receive a passivation film according to one embodiment of the present disclosure. The transmitting electric to acoustic wave transducer  110  and the receiving acoustic wave to electric transducer  110 ′ together define an interdigitated SAW transducer  105  as is known in the art. The transmitting electric to acoustic wave transducer  110  includes a first transmitting electrode comb-like or prong-like electrically conductive member  112  that interlocks or interdigitates with a second transmitting electrode comb-like electrically conductive member  122 . The first transmitting conductive member  112  includes a rectangularly-shaped base  114  from which extend orthogonally first rectangularly-shaped peripheral prong  116   a , rectangularly-shaped central prong  116   b  and second rectangularly-shaped peripheral prong  116   c.    
     As noted, the transmitting electric to acoustic wave transducer  110  also includes the second transmitting electrode comb-like or prong-like electrically conductive member  122  that interlocks or interdigitated with the first transmitting electrode comb-like electrically conductive member  112 . The second transmitting conductive member  122  includes a rectangularly-shaped base  124  from which extend orthogonally first rectangularly-shaped prong  126   a  and second rectangularly-shaped prong  126   b . The rectangularly shaped base  124  defines a first end  124   a  and a second end  124   h . The first prong  126   a  is positioned at a distance away from first end  124   a  and the second prong  1261  is positioned at a distance away from the second end  126   b  to define a central rectangularly-shaped aperture  128  between the first prong  126   a  and the second prong  126   b.    
     The first transmitting electrically conductive member  112  includes a voltage input terminal Vin that is in direct electrical communication with, for example, the rectangularly-shaped base  114  such that the entire first transmitting electrically conductive member  112  is in electrical communication with the voltage input terminal Vin. The first and second electrically conductive members  112  and  122  are arranged such that the prongs  116   a ,  116   b ,  116   c  are adjacent to and intermesh with prongs  126   a  and  126   h . The second transmitting electrically conductive member  122  includes a ground terminal  130  that is in direct electrical communication with, for example, the rectangularly-shaped base  124  such that the entire second transmitting electrically conductive member  122  is in electrical communication with the ground terminal  130 . 
     The receiving acoustic wave to electric transducer  110 ′ includes the same components as, and is arranged in the same manner as, transmitting electric to acoustic wave transducer  110  and for convenience the components are designated with primes. 
     Consequently, receiving acoustic wave to electric transducer  110 ′ includes a first transmitting electrode comb-like or prong-like electrically conductive member  112 ′ that interlocks or interdigitates with a second transmitting electrode comb-like electrically conductive member  122 ′. The first receiving electrically conductive member  112 ′ includes rectangularly-shaped base  114  from which extend orthogonally first rectangularly-shaped peripheral prong  116   a ′, rectangularly-shaped central prong  116   b ′ and second rectangularly-shaped peripheral prong  116   c′.    
     The receiving electric to acoustic wave transducer  110 ′ also includes the second receiving electrode comb-like or prong-like electrically conductive member  122 ′ that interlocks or interdigitates with the first receiving electrode comb-like electrically conductive member  112 ′. The second receiving conductive member  122 ′ includes a rectangularly-shaped base  124 ′ from which extend orthogonally first rectangularly-shaped prong  126   a ′ and second rectangularly-shaped prong  126   b ′. The rectangularly-shaped base  124 ′ defines a first end  124   a ′ and a second end  124   b ′. The first prong  126   a ′ is positioned at a distance away from first end  124   a ′ and the second prong  126   b ′ is positioned at a distance away from the second end  126   b ′ to define a central rectangularly-shaped aperture  128 ′ between the first prong  126   a ′ and the second prong  126   b′.    
     The first receiving electrically conductive member  112 ′ includes a voltage output terminal Vout that is in direct electrical communication with, for example, the rectangularly-shaped base  114 ′ such that the entire first receiving electrically conductive member  112 ′ is in electrical communication with the voltage output terminal Vout. The first and second electrically conductive members  112 ′ and  122 ′ are arranged such that the prongs  116   a ′,  116   b ′,  116   c ′ are adjacent to and intermesh with prongs  126   a ′ and  126   h ′. The second receiving electrically conductive member  122 ′ includes a ground terminal  130 ′ that is in direct electrical communication with, for example, the rectangularly-shaped base  124 ′ such that the entire second receiving electrically conductive member  122 ′ is in electrical communication with the ground terminal  130 ′. 
     When a voltage source, not shown, is placed across the terminals Vin and Vout, a surface acoustic wave  150  is generated by the transmitting electric to acoustic wave transducer  110  and travels in the direction of arrow  152  towards the receiving acoustic wave to electric transducer  110 ′. 
       FIG. 3  illustrates a cross-sectional view of a coated piezoelectric SAW transducer biofilm sensor  200  according to one embodiment of the present disclosure. More particularly, surface acoustic wave (SAW) biofilm sensor  200  includes the interdigitated SAW transducer  105  of  FIG. 2 . Accordingly, the SAW biofilm sensor  200  includes transmitting electric to acoustic wave transducer  110  defining an upper surface  110   a  and a lower surface  110   b  where the cross-section (not shown) is taken through the rectangularly-shaped base  124 . The surface acoustic wave (SAW) biofilm sensor  200  also includes receiving acoustic wave to electric transducer  110 ′ defining an upper surface  110   a ′ and a lower surface  110   b′.    
     The lower surface  110   a  of the transmitting electric to acoustic wave transducer  110  is disposed on a portion  301   a  of the upper surface  30   a  of the piezoelectric SAW loss reduction film layer  30  and the lower surface  110   a ′ of the receiving acoustic wave to electric transducer  110 ′ is disposed on another portion  302   a  of the upper surface  30   a  of the piezoelectric SAW loss reduction film layer  30 . 
     A piezoelectric film layer  160  defines an upper surface  160   a  and a lower surface  160   b . A portion  1601   b  of the lower surface  160   b  of the piezoelectric film layer  160  is disposed on the upper surface  110   a  of the transmitting electric to acoustic wave transducer  110 . Another portion  1602   b  of the lower surface  160   b  of the piezoelectric film layer  160  is disposed on the upper surface  110 ′ a  of the receiving acoustic wave to electric transducer  110 ′. 
     A passivation film layer  170  defines an upper surface  170   a  and a lower surface  170   b , The lower surface  170   b  of the passivation film layer  170  is disposed on the upper surface  160   a  of the piezoelectric film layer  160 . Thus, the upper surface  160   a  of the passivation film layer  170  is thereby configured to enable contact with a biofilm  40 . 
     In a similar manner as described with respect to  FIG. 1 , the coated piezoelectric SAW transducer biofilm sensor  200  may be disposed or mounted on transducer mounting structure  35  which include common piezoelectric loss reduction film layer  30  and substrate  32 . The substrate  32  defines an upper surface  32   a  and a lower surface  32   b . Similarly, the common piezoelectric loss reduction film layer  30  defines the upper surface  30   a  and a lower surface  30   b . The lower surface  30   b  of the piezoelectric SAW loss reduction film layer  3030  is disposed on the upper surface  32   a  of the substrate  32 . 
     A portion  1603   b  of the lower surface  160   b  of the piezoelectric film layer  160  is disposed on a portion  303   a  of the upper surface  30   a  of the piezoelectric SAW loss reduction film layer  30  and disposed between the transmitting electric to acoustic wave transducer  110  and the receiving acoustic wave to electric transducer  110 ′. 
       FIG. 4A  illustrates a top perspective schematic view of the exemplary embodiment of the SAW biofilm sensor  200  of  FIGS. 2 and 3  according to one embodiment of the present disclosure that includes the piezoelectric SAW transducer  105  further including the piezoelectric layer  160  and the passivation layer  170  disposed on the upper surfaces  110   a  and  110   a ′ of the SAW transducer  105  and further illustrating a SAW  150  passing in the direction of arrow  152  through a biofilm  40  on the upper surface  170   a  of the passivation layer  170 . 
       FIG. 4B  illustrates a cross-sectional schematic view of the SAW biofilm sensor  200  of  FIG. 4A  illustrating the piezoelectric transducer  105  and the thickness dimensions of the piezoelectric layer  160 , the passivation layer  170  and the biofilm  40 . More particularly, in the exemplary embodiment of  FIG. 4B , the piezoelectric layer  160  may define an upper sub-layer  162  and a lower sub-layer  161  which together define thickness d of the piezoelectric layer  160 . 
     The lower sub-layer  161  is defined by the portion  1603   b  of the lower surface  160   b  of the piezoelectric film layer  160  that is disposed on the portion  303   a  of the upper surface  30   a  of the lower piezoelectric film layer  30  and is disposed between the transmitting electric to acoustic wave transducer  110  and the receiving acoustic wave to electric transducer  110 ′. The upper sub-layer  162  is defined by the portion of the piezoelectric layer  160  between the passivation film layer  170  and the lower sub-layer  161  of the piezoelectric layer. 
     The lower sub-layer  161  has a shear modulus and density to define a first SAW velocity V1. The upper sub-layer  162  has a shear modulus and density to define a second SAW velocity V2. Although the second velocity V2 may differ from the first velocity V1, for the purposes of simplifying the design analysis, the two velocities may be set equal to one another. Alternatively, the second velocity V2 may be equal to or greater than the first velocity V1. 
     The passivation layer  170  defines a thickness h between the upper surface  160   a  of the passivation layer  160  and the lower surface  160   b  of the passivation layer  160  while the biofilm  40  defines a thickness x. 
     In one embodiment, the piezoelectric layer  160  includes zinc oxide, ZnO and the thickness d of the piezoelectric layer  160  has a dimension of at least 40 nanometers (nm). 
     In one embodiment, the passivation layer includes aluminum oxide, Al 2 O 3 , and the thickness h of the passivation layer  170  has a dimension of at least 45 nanometers (nm). 
     The specific design for the selection of the material and thickness of the piezoelectric layer  160  and the passivation layer  170  is described in more detail below. 
     Materials and Methods 
     Design of the SAW Sensor 
     For applications of a SAW sensor in liquid environments, selecting the proper mode of propagation is crucial to prevent severe attenuation or the wave. In a SAW sensor, the surface of the piezoelectric layers is set to a high frequency oscillation governed by the design of the interdigitated transducers (IDT) and the SAW velocity of the piezoelectric material. This no-load oscillation frequency is affected by environmental changes at the surface of the SAW sensor. These effects are observed experimentally as changes in resonant frequency, representing a shift in the SAW phase velocity. However, one of the challenges for biosensor applications is the extremely high attenuation damping of the SAW in liquid environments, when Rayleigh mode waves are generated. In this mode the acoustic wave displacement is perpendicular to the surface and causes significant attenuation of the oscillations in liquid environments. Unlike Rayleigh mode waves, Love mode SAW generation demonstrates displacement planar to the surface and the oscillations are not attenuated in liquid environments [11-15, 18-20, 29-31]. The generation of Love or Rayleigh mode waves depends on the crystallographic orientation of the piezoelectric film. Therefore, it is highly advantageous to deposit piezoelectric material with a specific orientation for generating Love mode SAW [16-22, 29-31]. ZnO with a high piezoelectric coefficient is capable of generating very high frequency (GHz, gigahertz, where Hz is hertz or 1 cycle/sec) SAW, and it has been shown to grow along the crystallographic orientation that favors Love mode propagation on a SiO 2 /Si substrate [19-22]. Love mode waves are predominantly generated with the SAW IDT aligned perpendicular to the c-axis of the ZnO film [16-22]. 
     Some important parameters, such as the SiO.sub.2 thickness, the IDT electrode dimensions, and the ZnO deposition method, had to be considered in the design of a highly sensitive SAW sensor. In order to confine the propagation of the SAW on the surface of the device, a thin film that can prevent acoustic wave loss from the piezoelectric material to Si substrate was required between ZnO film and Si. SiO 2  was previously shown [15, 18] to be an appropriate loss blocking film for thicknesses around 50 nm and was selected in this work. For the best resonance of the SAW at the designed operational frequency, the IDT separation d should be equal to half of the operational wavelength λ. (See  FIG. 7B ). The acoustic wave velocity for the ZnO thin film used in this work was 4814 m/s, and the operational frequency (401 MHz-406 MHz) of the SAW sensor was designed to meet the regulation set by the Federal Communication Commission (FCC) for future biomedical biofilm detection applications [32, 48]. This wavelength (λ) was 12 μm, rendering the electrode separation of the IDT 6 μm (λ/2). The crystal quality of ZnO film also had to be high for a sensitive SAW sensor. The ZnO film with low impurities and lattice defects was achieved by PLD which has been widely used in metal oxides for high quality film deposition due to stoichiometric deposition with the target material [33] and a relatively simple set-up. 
     To achieve advantages over prior art biofilm sensors for biofilm detection, it was necessary to provide an inverted structure for the saw sensor  200 , as shown in  FIGS. 3, 4A and 4B  as compared to the structure of a standard SAW sensor such as biofilm sensor  20  as shown in  FIG. 1 . In traditional SAW sensors such as biofilm sensor  20 , the IDT is exposed to the liquid environment directly, causing IDT corrosion in long term studies. However, in the inverted SAW sensor  200  according to  FIGS. 3, 4A and 4B , the IDT lifetime is extended because the IDT is patterned under the piezoelectric film. The sensitivity of the Love mode SAW sensor in the top and bottom of piezoelectric films also show the same level of sensitivity for our designed ZnO film thickness (400 nm) based on the Love mode propagation depth [11-14, 29]. 
     The material of the IDT, traditionally aluminum in the SAW sensor, can be selected by the acoustic impedance match theory [34]. Potential materials, such as aluminum and gold, were selected and the acoustic power reflective coefficient (R) was calculated based on the theory. The reflective coefficient of the aluminum and gold were 0.058 and 0.012 respectively. The lower reflective coefficient in IDT represents more energy transmission to the piezoelectric material which makes a highly sensitive SAW sensor. Therefore, the IDT material was chosen to be gold based on the low R value. 
     Selection of Passivation Film 
     Since the bare ZnO layer  160  without a passivation film layer  170  was damaged both in LB media and 10% FBS, selection of the proper material to protect ZnO while considering future biomedical applications is important to maintain the sensitivity of the sensor. The sensitivity of the passivated SAW sensor is decreased as compared to the unpassivated sensor due to the initial mass loading and dispersion of the wave in the passivation film [12, 29, 35-40]. To investigate the effect of the added material on the SAW sensitivity in addition to the material selection, we consider only the mass loading effect of the passivation film based on the assumption that the dispersion in the passivation film is minimal due to a much thinner passivation layer (45 nm) as compared to the wavelength of the SAW [35, 38, 40]. As noted, the schematic cross section view of the inverted passivation SAW sensor  200  is shown in  FIGS. 3, 4A and 4B . The sensitivity of the SAW sensor (S m   v ) is directly proportional to the velocity change due to the mass loading as shown in equation (1) [15, 18-20]. 
     
       
         
           
             
               
                 
                   
                     S 
                     m 
                     v 
                   
                   = 
                   
                     
                       lim 
                       
                         
                           Δ 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           m 
                         
                         -&gt; 
                         0 
                       
                     
                     ⁢ 
                     
                       
                         1 
                         
                           Δ 
                           ⁢ 
                           
                               
                           
                           ⁢ 
                           m 
                         
                       
                       ⁢ 
                       
                         ( 
                         
                           
                             Δ 
                             ⁢ 
                             
                                 
                             
                             ⁢ 
                             v 
                           
                           
                             v 
                             0 
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 
                   ( 
                   1 
                   ) 
                 
               
             
           
         
       
     
     where v 0  is the initial velocity of the wave, m is the amount of the additional mass, and Δv is the wave velocity changes due to Δm. The SAW velocity (v), shown in equation (2), is defined by the shear modulus of the piezoelectric material and local area density based on the one-dimensional acoustic wave equation (3) [15, 18-20]. 
     
       
         
           
             
               
                 
                   v 
                   = 
                   
                     
                       C 
                       p 
                     
                   
                 
               
               
                 
                   ( 
                   2 
                   ) 
                 
               
             
             
               
                 
                   
                     
                       
                         ∂ 
                         2 
                       
                       ⁢ 
                       u 
                     
                     
                       ∂ 
                       
                         t 
                         2 
                       
                     
                   
                   = 
                   
                     
                       ( 
                       
                         C 
                         ρ 
                       
                       ) 
                     
                     ⁢ 
                     
                       
                         
                           ∂ 
                           2 
                         
                         ⁢ 
                         u 
                       
                       
                         ∂ 
                         
                           y 
                           2 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   3 
                   ) 
                 
               
             
           
         
       
     
     where C is the shear modulus of the surface material, u is the mechanical displacement of the piezoelectric substrate, ρ is material density of the surface, y is the axis of the mechanical displacement propagation, and v is the velocity of the SAW in equations (2) and (3). In order to simplify the modeling of passivation effects on the sensitivity of the SAW sensor, the bacterial growth over the sensor was assumed uniform, so that bacterial mass loading only depended on the thickness of the biofilm. Based on this assumption, the sensitivity of the SAW sensor from equation (1) was proportional to the velocity change as biofilm thickness (x) approaches zero as shown in equation (4). 
     
       
         
           
             
               
                 
                   
                     S 
                     m 
                     v 
                   
                   ∝ 
                   
                     
                       ⅆ 
                       v 
                     
                     
                       ⅆ 
                       
                         x 
                         
                           x 
                           -&gt; 
                           0 
                         
                       
                     
                   
                 
               
               
                 
                   ( 
                   4 
                   ) 
                 
               
             
           
         
       
     
     The biofilm formed on the sensor also has a comparatively low shear modulus that can be neglected in the calculation of the total shear modulus of the passivated SAW sensor. The total shear modulus including the ZnO and the passivation film was calculated based on a mechanical spring series connection because the SAW is transferred from ZnO film to the passivation layer sequentially. Based on the assumptions and the total shear modulus calculation, the SAW velocity on a sensor coated with a biofilm was determined by the following equation (5). 
     
       
         
           
             
               
                 
                   v 
                   = 
                   
                     
                       
                         
                           C 
                           ZnO 
                         
                         
                           
                             ρ 
                             ZnO 
                           
                           ⁢ 
                           d 
                         
                       
                       ⁢ 
                       
                         ( 
                         
                           
                             C 
                             film 
                           
                           
                             
                               C 
                               ZnO 
                             
                             + 
                             
                               C 
                               film 
                             
                           
                         
                         ) 
                       
                       ⁢ 
                       
                         ( 
                         
                           1 
                           
                             1 
                             + 
                             
                               
                                 
                                   ρ 
                                   film 
                                 
                                 ⁢ 
                                 h 
                               
                               
                                 
                                   ρ 
                                   ZnO 
                                 
                                 ⁢ 
                                 d 
                               
                             
                             + 
                             
                               
                                 
                                   ρ 
                                   bac 
                                 
                                 ⁢ 
                                 x 
                               
                               
                                 ρ 
                                 ZnO 
                               
                             
                           
                         
                         ) 
                       
                     
                   
                 
               
               
                 
                   ( 
                   5 
                   ) 
                 
               
             
           
         
       
     
     where C film  and C Zno  are the shear moduli of the passivation film and ZnO, ρ Zno  and ρ film  are the densities of the ZnO and passivation film, h is the thickness of the passivation film, d is the thickness of the ZnO film, and x is the biofilm thickness. All parameters except the biofilm thickness (x) were determined by selecting potential passivation materials (i.e. Al 2 O 3 , Si 3 N 4 , SiO 2  and Teflon) and their thicknesses which were assumed to be 40 nm for all passivation films considered. Potential passivation materials with mechanical properties similar to the ZnO film, such as shear modulus and density, were selected [41, 42]. The sensitivity of the SAW sensor with different passivation films was calculated by differentiating equation (5) with respect to x, and letting x approach zero, based on equation (4). 
       FIG. 8  graphically illustrates the normalized theoretical sensitivity for, from left to right, without passivation, aluminum oxide —Al 2 O 3 , silicon nitride —Si 3 N 4 , silicon dioxide —SiO 2 , and Teflon. The lowest degradation in sensitivity is observed for the Al 2 O 3  passivation layer with a 0.20 reduction, while Teflon had the maximum degradation at 0.95 reduction. 
     The application of this fundamental theoretical treatment showed that an Al 2 O 3  film was best suited as a passivation layer and it was selected. The thickness of the passivation film (h) in equation (5) is important for our application. The film should be thick enough for effective passivation, but should not be too thick that the added layer causes a significant attenuation both due to mass loading and dispersion of the SAW resulting in substantial loss in sensitivity. The minimum required thickness of Al 2 O 1  film (45 nm) was empirically determined. The detailed studies were presented in the film characterization section. 
     Fabrication 
     As described below in more detail with respect to  FIG. 5 , to create the piezoelectric layer  160  on the transducer mounting structure  35 , the target material employed herein is zinc oxide, ZnO. Stoichiometric film deposition is achieved by application of a 248 nm KrF laser in 25 ns pulses at 250° C. 
       FIG. 6  illustrates a schematic diagram of atomic layer deposition of the passivation layer  170  of  FIGS. 3, 4A and 4B . 
     The fabrication process flow is shown in  FIGS. 7A through 7E . Referring first to  FIG. 7A , a silicon dioxide (SiO 2 ) layer  30  was deposited on the upper surfaces  32   a  of (100) Si substrates  32  by low pressure chemical vapor deposition (LPCVD) as studied in [15, 22]. 
     Referring to  FIG. 7B , the IDT  105  was patterned using traditional photolithography before depositing the piezoelectric layer  160 , e.g., ZnO film. The IDT prongs  116   a  and  116   a ′ are deposited to have a width W1 of 1 μm (microns), prongs  116   b  and  116   b ′ are deposited to have a width W2 of 1.5 μm, and prongs  116   c  and  116   c ′ are deposited to have a width W3 of 2 μm. Due to the small feature size of the IDT  105 , including 1 μm, 1.5 μm, and 2 μm wide electrodes, oxygen plasma was used to remove residual photoresist after development. 
     In  FIG. 7C , Cr/Au (15 nm/200 nm) as the IDT  105  material was deposited on the wafer or transducer mounting structure  35  by E-beam evaporation, followed by lift-off. The transducer mounting structure  35  was diced into smaller segments before deposition of the piezoelectric layer  160 , composed of ZnO, was performed by pulsed laser deposition (PLD). Crystalline [001] orientation ZnO films on SiO 2 /(100)Si substrates were grown by PLD. The piezoelectric layer  160  did not entirely cover the IDT  105  such that an indentation  162   a  from longitudinal external edge  1161   a  of the prong  116   a  and an indentation  162   b  from longitudinal external edge  1162   c ′ of the prong  116   c ′ were formed in the piezoelectric layer  160  to subsequently accommodate portions of the passivation layer  170 . 
       FIG. 7D  illustrates the annealing process of the ZnO film pattern wherein the ZnO film pattern was annealed at 800° C. for 1 hour. 
       FIG. 7E  is a schematic diagram of passivation layer  170  deposited partially on the electrode pattern  105  of  FIGS. 7B, 7C and 7D  and on the piezoelectric layer  160  of  FIGS. 7C and 7D . Vertical edges  172   a ,  172   b  of the passivation layer  170  are formed by the atomic layer deposition to fill the indentations  162   a ,  162   b , respectively, of the piezoelectric layer  160 . 
     For simplicity, in  FIGS. 7B to 7E , only the prongs  116   a ,  116   b ,  116   c  of the first transmitting electrically conductive member  112  and only the prongs  116   a ′,  116   b ′,  116   c ′ of the first receiving electrically conductive member  112 ′ as shown in  FIG. 2  are illustrated. The second transmitting electrically conductive member  122  and the second receiving electrically conductive member  122 ′ are omitted in  FIGS. 7B to 7E . 
     Returning to  FIG. 5 , there is illustrated a schematic diagram of a pulsed layer deposition (PLD) system for the piezoelectric layer  160  on the transducer mounting structure  35  that includes common piezoelectric loss reduction layer  30  and substrate  32  for assembling the piezoelectric SAW biofilm sensor  200  of  FIGS. 2, 3, 4A and 4B . More particularly, pulsed laser deposition (PLD) system  300  includes a vacuum chamber  302  having a rotating target carousel  304 . The carousel is rotatable by a first shaft  306  which passes through a first penetration  308  in the vacuum chamber  302 . The shaft  306  rotates a platform  310  supported on the target carousel  304  and supports the target  312  which is thus capable of being rotated by the first shaft  306 . 
     The transducer mounting structure  35  is positioned directly opposite to the target  312  on the target platform  310  and the target carousel  304 . The structure  35  is positioned on a heatable sample stage  314  that is supported by a second shaft  316  that passes through a second penetration  318  in the vacuum chamber  302 . 
     A quartz window  320  is positioned in the vacuum chamber  302  at an angle to enable a beam of laser light  324  to impact the target  312 . As a result of the beam of laser light  324  impacting the target  312 , a plume  326  of target material is created within the vacuum chamber and deposits on the transducer mounting structure  35  which includes substrate  32 . 
     The laser deposition system used a KrF excimer laser at a wavelength of 248 nm with pulse duration of 25 ns to ablate a high purity (99.99%) ZnO ceramic target. The ZnO layer was grown at 250° C. with an ambient oxygen partial pressure of ˜1.0×10 −4  Torr. After ZnO film deposition, electrical contact pad areas were patterned by photolithography and the ZnO was etched using a solution that consisted of phosphoric acid, acetic acid, and deionized water (1:1:30). 
     As shown in  FIG. 7D , the partially completed biofilm sensor  200  was annealed at 800° C. for one hour to increase the resistivity of the ZnO [43]. After annealing, the resistance measured in the IDT increased from 150Ω to 30-40 MΩ. As compared to  FIG. 7C , there are generally no dimensional changes for the piezoelectric layer  160 , the electrode pattern  105  or the transducer mounting structure  35  as a result of the annealing process. 
       FIG. 6  is a schematic illustration of a gas-based flow ALD process  400  to cause the passivation.  FIG. 7E  is a schematic diagram of the passivation layer  170  deposited partially on the electrode pattern of  FIGS. 7B, 7C and 7D  and on the piezoelectric layer  160  of  FIGS. 7C and 7D . Referring now to  FIG. 6  and  FIG. 7E , the ZnO surface  160   a  of the SAW sensor  200  may be passivated by depositing an Al 2 O 3  film using atomic layer deposition (ALD. To deposit the passivation layer  170 , Al 2 O 3  ALD thin films were fabricated at 150° C. in a flow-through chamber of a Beneq Model TFS-500 atomic layer deposition reactor system (available from Beneq Oy, Vantaa, Finland). As shown on the left side of  FIG. 6 , trimethylaluminum (TMA) Al 2 (CH 3 ) 3 , originating as a liquid precursor (not shown) that is converted to a vapor at  402  was introduced into the ALD reactor (not shown) and formed a single layer of the TMA  404  on the ZnO layer  160 . In the center of  FIG. 6 , the single layer of the TMA  404  is purged of unbound TMA vapor to become a purged single layer  406  of the TMA. As shown on the right side of  FIG. 6 , water vapor (H 2 O)  414  as the oxygen source was introduced into the ALD reactor and a covalent bonding between the TMA layer  406  and hydroxyl group of the water vapor  414  was established creating a single atomic layer of aluminum oxide  416  deposition. Each deposition cycle results in a 0.09 nm/cycle of deposition rate consistently. The single atomic layer of aluminum oxide Al 2 O 3    416  thus becomes the passivation layer  170 . To illustrate that the TMA layer  406  has bonded with the hydroxyl group of the water vapor  414 , the size of the circles representing the aluminum oxide Al 2 O 3    416  are shown to be larger than the size of the circles representing the TMA layer  406 . 
     In view of the foregoing, those skilled in the art will understand that embodiments of the present disclosure relate to a surface acoustic wave (SAW) biofilm sensor  200  that includes SAW transducer  105 , piezoelectric film layer  160 , and passivation film layer  170 . The piezoelectric film layer  160  is mounted over the SAW transducer  105  and the passivation film layer  170  is mounted over the piezoelectric film layer  160 . In one embodiment, the passivation layer  170  includes aluminum oxide, Al 2 O 3 . In yet another embodiment, the passivation layer  170  defines a thickness h of at least nanometers (nm). In a still further embodiment, the piezoelectric layer  160  includes zinc oxide, ZnO. In yet another embodiment, the piezoelectric layer  160  defines a thickness d of at least 40 nanometers (nm). 
     Further, in view of the foregoing, those skilled in the art will understand that embodiments of the present disclosure relate to a method of assembling a biofilm surface acoustic wave (SAW) sensor, e.g., biofilm SAW sensor  200 , that includes depositing a piezoelectric layer, e.g., piezoelectric layer  160 , on a SAW transducer electrode pattern, e.g., IDT electrode pattern  105 , and depositing a passivation layer, e.g., passivation layer  170 , on the piezoelectric layer  160 . The depositing a piezoelectric layer may include depositing a layer of zinc oxide ZnO on the SAW transducer electrode pattern  105 . In one embodiment, the depositing a layer of zinc oxide ZnO on the SAW transducer electrode pattern  105  may include depositing a layer of zinc oxide ZnO having a thickness d of at least 40 nanometers (nm). 
     Further, the depositing a passivation layer, e.g., passivation layer  170 , on the piezoelectric layer, e.g., piezoelectric layer  160 , may include depositing a layer of aluminum oxide Al 2 O 3  on the piezoelectric layer  160 . The depositing a layer of aluminum oxide Al 2 O 3  on the piezoelectric layer includes depositing a layer of aluminum oxide Al 2 O 3  having a thickness h of at least 45 nanometers (nm) on the piezoelectric layer. 
     Additionally, the method of assembling the SAW biofilm sensor  200  may further include depositing the SAW transducer electrode pattern, e.g., IDT electrode pattern  105 , on a piezoelectric SAW loss reduction film layer, e.g., piezoelectric SAW loss reduction film layer  30 . 
     The method of assembling may further include depositing the piezoelectric SAW loss reduction film layer, e.g., piezoelectric. SAW loss reduction film layer  30 , on a substrate, e.g. substrate  32 . 
     The step of depositing a piezoelectric layer, e.g., piezoelectric layer  160 , on a SAW transducer electrode pattern, e.g., IDT electrode pattern  105 , may include pulsed laser deposition. Additionally, the step of depositing a passivation layer, e.g., passivation layer  170 , on a piezoelectric layer, e.g., piezoelectric layer  160 , may include atomic layer deposition. 
     Device Characterization and Testing 
     Before the SAW sensor was used to measure biofilm growth, the performance of the passivation film was characterized using an optical microscope to inspect the surface of the ZnO layer after exposure to growth media. The results were used to optimize the film thickness and fabrication process. The sensitivity of the sensor was studied by loading the sensor surface with &amp;ionized (DI) water since its viscosity is negligible. After these characterization studies, the sensor response was tested using  E. coli  static biofilm growth. 
     Al 2 O 3  Film Characterization 
     Based on the theoretical modeling calculation presented previously and the results shown in  FIG. 8 , Al 2 O 3  was selected as a passivation film. Al 2 O 3  films were deposited to thicknesses of 20 nm-45 nm by ALD to investigate the minimum required thickness for ZnO passivation. SAW sensors with four different thicknesses (20 nm, 30 nm, 40 nm, and 45 nm) of ALD Al 2 O 3  film were placed in a LB media bacterial suspension for two days. The surface of the device was inspected using optical microscopy. As shown in  FIGS. 9-12 , visible ZnO damage was observed when the thickness of ALD Al 2 O 3  was thinner than 45 nm. 
     More particularly,  FIG. 9  is an optical microscopy image of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 20 nm showing damaged areas.  FIG. 10  is an optical microscopy image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 30 nm showing damaged areas.  FIG. 11  is an optical microscopy image of yet another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 40 nm showing damaged areas. Finally,  FIG. 12  is an optical microscopy image of another SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  passivated by ALD of Al 2 O 3  with a passivation layer thickness of 45 nm showing no damage. 
     Based on these experiments, the minimum required thickness of ALD Al 2 O 3  film for the passivation of ZnO was 45 nm. Since thicker passivation films caused a high loss of sensitivity due to more initial mass loading, the 45 nm thick Al 2 O 3  film was selected to passivate the SAW sensor. 
     In addition to ALD, other Al 2 O 3  film deposition methods were investigated in order to evaluate the dependence of passivation layer performance on the fabrication process. E-beam evaporation and RF-sputtering were used to deposit 45 nm of Al 2 O 3  film. However, after two days in an LB media bacterial suspension, these two passivation films were not able to protect the ZnO layer as shown in  FIGS. 13-15 . 
     More particularly,  FIG. 13  is an optical surface image of another SAW biofilm sensor  200  assembled in accordance with  FIGS. 2-7E  passivated by a 45 nm Al 2 O 3  film using e-beam evaporation showing damage.  FIG. 14  is an optical surface image of still another SAW biofilm sensor  200  assembled in accordance with  FIGS. 2-7E  passivated by a 45 nm Al 2 O 3  film using RF sputtering showing damage.  FIG. 15  is an optical surface image of yet another SAW biofilm sensor  200  assembled in accordance with  FIGS. 2-7E  passivated by a 45 nm Al 2 O 3  film using ALD showing no damage. 
     This result can be due to non-uniform or lower density film deposition of IE-beam evaporation and RF-sputtering as compared to ALD. Therefore, ALD is a highly advantageous fabrication process for effective passivation of the ZnO using Al 2 O 3 . 
     Passivated SAW Sensor Characterization 
     The mass sensitivity and detection limit of the SAW sensor were studied by loading 10 μl of deionized (DI) water onto the sensor. A volume of 10 μl was used since that was the minimum volume of DI water required to cover the area between the two IDTs of the sensor. By measuring the resonant frequency shift upon mass loading, the sensitivity of the SAW sensor was calculated based on equations (3) and (6). The mass detection limit of the sensor was also calculated using the equipment resolution and the sensitivity. 
     Real-Time Resonant Frequency Monitoring 
     As illustrated in  FIG. 16 , for real time resonant frequency monitoring in bacterial biofilm formation experiments, a custom biofilm sensing package  500  was designed to enable low impedance Bayonet Neill-Concelman (BNC) cable connections for RF applications with a network analyzer (HP8510B, Agilent Inc. USA) (not shown). The device biofilm sensing package  500  includes a bacterial growth well  502 , which was used to prevent bacterial growth media leakage and localize the bacterial growth, and a chip package  504 , that includes two separate portions  5041  and  5042  on either longitudinal side of the SAW sensor  200  connecting the sensor input BNC cable  510  and sensor output BNC cable  512  and BNC cables  520  and  522  for generating RF radiation. The SAW sensor  200  was placed in the bacterial growth well  502  and connected to the BNC connectors  520  and  522  by lead soldering on the chip package  504 . The network analyzer was used to sweep a wide range of RF frequencies into the sensor  200  and the device resonant frequency was analyzed using S-parameter analysis. The resonant frequency of the sensor  200  was detected by measuring a low peak of the reflective power ratio (S 11 ) in the network analyzer. Data was collected and saved to a computer (not shown) every minute using general purpose interface bus (GPIB) communication (not shown) with the network analyzer. 
     Biofilm Growth Experiments with the SAW Sensor 
       E. coli  W3110 was cultured in a shaking incubator for about 16 hours. The grown bacterial suspension was diluted with LB media or 10% FBS to make the initial OD 600  approximately 0.21-0.23. The total volume of the diluted bacterial suspension in the growth well  502  was 20 ml in experiments with both types of media. The FBS solution was prepared to a 10% concentration by diluting with Dulbecco/Vogt modified Eagle&#39;s minimal essential medium (Invitrogen Inc, USA). After filling the bacterial growth well  502  with the diluted bacterial suspension, the well was sealed by paraffin film to prevent evaporation of the media during the experiment. The package  500  was placed on a 37° C. hotplate  530 , and a polystyrene container (not shown) covered the whole package to reduce the temperature gradient near the test setup. After each biofilm growth experiment, the sensor  200  was recalibrated using DI water loading. The thickness of biofilm was measured optically by the distance difference between the focal plane of the sensor surface and the focal plane of the top of any accumulated biofilm. 
     Results and Discussion 
     ZnO Film Characterization 
     The generation of Love mode SAW was confirmed by investigating the lattice orientation of the deposited ZnO thin film, X-ray diffraction (XRD) was employed for crystal structure characterization of the ZnO layer after PLD deposition on a SiO 2 /Si substrate by measuring the diffraction angle (2θ). The diffraction angles of the in the ZnO film at 34.25° and 72.25°, corresponding to c-axis (002) and (004) lattice orientations, were the most intensive reflections in the PLD prepared ZnO film. This c-axis orientation of ZnO crystal lattice ((00L) direction) was perpendicular to the substrate so that the Love mode of SAW generation was dominant on the surface of the sensor [15-22]. 
     Photoluminescence (PL) spectroscopy was used to investigate the crystal quality of the ZnO film. The peak wavelength of the emitted light was approximately 380 nm, corresponding to the characteristic ZnO bandgap energy (3.3 eV). Therefore, the PL spectroscopy result confirmed that the PLD-prepared ZnO film had a low number of impurities. 
     Biofilm Cleaning 
     Consecutive biofilm growth tests using the same device are essential to investigate the reliability and repeatable operation of the SAW sensor. To test the sensor over multiple biofilm growth experiments, surface cleaning after a biofilm growth experiment was crucial for subsequent biofilm growth; cleaning not only sterilized the sensor, but also prevented initial mass loading due to the uncleaned biofilm and the resulting significant loss of sensitivity. Oxygen plasma applied for 30 s at 150 W RF-power was successfully employed to clean any remaining biofilm as shown in  FIGS. 17 and 18   
       FIG. 17  is an optical microscopy image of the surface of the SAW biofilm sensor  200  assembled in accordance with  FIGS. 2-7E  before biofilm cleaning. 
       FIG. 18  is an optical microscopy image of the surface of the SAW biofilm sensor  200  assembled in accordance with  FIGS. 2-7E  after oxygen plasma biofilm cleaning. 
     The 45 nm ALD Al 2 O 3  film passivation and oxygen plasma biofilm cleaning method enable SAW sensor  200  to be reusable over consecutive biofilm growth experiments. 
     Sensor Sensitivity 
     The sensitivity and detection limit of the SAW sensor were studied and calculated by loading 10 μl of DI water on the sensor and monitoring the magnitude of the resonant frequency shift. After loading 10 si of DI water, the resonant frequency shift of the SAW sensor was measured to be about 188 KHz by the network analyzer. Hence, the sensitivity of the sensor was 1.88×10 10  Hz/g. Based on the network analyzer resolution (0.1 Hz), the detection limit of the sensor (resolution/sensitivity) was approximately 5.3 pg (picograms). Since the mass of a bacterium is known to be approximately 1 pg [44], this detection limit validates the SAW sensor application for bacterial biofilm monitoring. 
     Biofilm Growth Experiments in the SAW Sensor 
     The resonant frequency shift results of the SAW sensor due to the biofilm growth in LB media and in 10% PBS are shown in  FIGS. 19, 20 and 21 . 
       FIG. 19  is a graphical plot of negative resonant frequency shift results the SAW biofilm sensors assembled in accordance with  FIGS. 2-7E  versus time due to lysogeny broth (LB) biofilm growth for three newly fabricated SAW transducers. 
       FIG. 20  is a graphical plot of negative resonant frequency shift results for a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  versus time due to lysogeny broth biofilm growth in three sequential growth experiments using one device in an LB. 
       FIG. 21  is a graphical plot of negative resonant frequency shift results for a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  versus time in three growth experiments using a newly fabricated sensor in 10% fetal bovine serum (FBS). 
     In nature, bacterial growth in hatch culture begins with a lag phase where bacteria are not dividing but are actively adapting to the culture conditions. After this period, bacteria divide at a fast rate during the exponential phase of growth. Eventually, once the reactor contains a high population density and a limited supply of nutrients, the culture reaches stationary phase [45]. 
       FIG. 19  shows the resonant frequency shift results from three newly fabricated devices (device 1, 2, and 3) in the first biofilm growth experiment in LB media. The frequency shift results of each new sensor show exponential resonant frequency changes at the beginning without the preceding lag phase as compared to the natural bacterial growth trend. These output responses of the SAW sensor can be because the overnight cultured  E. coli  are in a metabolically active state. When active bacteria are diluted to the same growth media, the lag phase may not be observed because bacteria do not have to change their metabolism [46]. However, the stationary phase frequency shifts in LB media biofilm experiments are varied, i.e. 0.8 MHz, 2.3 MHz, and 4.1 MHz for each device. 
       FIG. 20  illustrates the results when one of the newly fabricated SAW sensors (device 2) after the first experiment of  FIG. 19  was selected and used for two additional sequential biofilm growth experiments, i.e., (2nd) and (3rd) in LB media, compared to the biofilm growth experiment for Device 2 in  FIG. 19 , designated as (1st), with the oxygen plasma cleaning applied between uses. The frequency shift results of sequential biofilm growth experiments in LB media as shown in  FIG. 20  correspond to an exponential growth at the beginning, but the frequency shift observed during stationary phase also varies from 1 MHz to 2.3 MHz. The detection limit of the SAW sensor after each sequential biofilm growth experiment was studied by loading 10 μl DI water on the sensor. The summary of the calculated detection limit of the SAW sensor for the consecutive experiments are shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Summary of the detection limit of the SAW sensor in sequential biofilm growth experiments. 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Before 
                 After 1 st   
                 After 2 nd   
                 After 3 rd   
               
               
                   
                 biofilm 
                 biofilm growth 
                 biofilm growth 
                 biofilm growth 
               
               
                   
                 experiment 
                 experiment 
                 experiment 
                 experiment 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Frequency shift due 
                 185 
                 kHz 
                 157 
                 kHz 
                 141 
                 kHz 
                 143 
                 kHz 
               
               
                 to 10 μl DI water 
               
               
                 Sensitivity 
                 1.85 × 10 10   
                 Hz/g 
                 1.57 × 10 10   
                 Hz/g 
                 1.41 × 10 10   
                 Hz/g 
                 1.43 × 10 10   
                 Hz/g 
               
               
                 Detection limit 
                 5.4 
                 pg 
                 6.4 
                 pg 
                 7.1 
                 pg 
                 7.0 
                 pg 
               
               
                   
               
            
           
         
       
     
     As shown in Table 1, the detection limit of the sensor changed minimally after consecutive biofilm growth experiments, demonstrating excellent sensitivity recovery of the sensor. Therefore, the large variance in the final resonant frequency shift seen in LB media is not a result of sensor degradation, but can be due to non-uniform growth of the biofilm based on the results shown in  FIGS. 19 and 20  and in Table 1. To investigate the biofilm growth variance in LB media and in 10% FBS, twelve test devices were prepared and placed in a bacterial suspension prepared as previously described. The biofilm thickness was measured after two days at 30 to 40 locations on the devices using an optical microscope. 
       FIG. 22  is a graphical plot of averaged biofilm thickness and standard deviation in LB media and in 10% FBS over 30-40 locations measured by optical microscopy. As shown in  FIG. 22 , the measured average biofilm thicknesses in LB media and in 10% FBS were 135 μm and 32 μm respectively. 
     The standard deviation of the measured biofilm thickness in LB media (62 μm, about 46% of the average biofilm thickness) was significantly more than the standard deviation in 10% FBS (6.4 μm, about 20% of average biofilm thickness). 
     These results correspond to the large growth variance in LB media measured by the resonant frequency shift of the SAW sensor. Based on these experimental biofilm growth variance results and the sensitivity characterization work as shown in Table 1, the stationary phase resonant frequency shift variation in LB media in  FIGS. 19 and 20  can be attributed to the natural variation in biofilm growth. The LB media, which is a standard bacterial growth media, is composed of essential materials for  E. coli  growth, such as amino acids, yeast, and NaCl. Thus, the media can provide a favorable environment for biofilm growth. The 10% FBS is mainly composed of diverse blood proteins, such as globular protein and Bovine Serum Albumin (BSA), and has been widely used as a simulated in vivo condition for mammalian cell culture. These composition differences between in LB media and in 10% FBS will cause different bacterial growth rates in each media, contributing to the observed difference in biofilm thickness. 
       FIG. 21  illustrates the resonant frequency shift results in 10% FBS biofilm growth experiments. In 10% FBS tests, a newly fabricated SAW sensor was used in three consecutive bacterial biofilm growth experiments, using oxygen plasma cleaning between experiments. As shown in  FIG. 21 , the frequency shift of the sensor also corresponds to exponential growth trend. Furthermore, the variation in the stationary phase in each experiment was only about 0.3 MHz which was much less than variation in LB media (about 3.3 MHz). This smaller difference in the final frequency shifts in 10% FBS compared to the LB media biofilm growth experiments can be due to the more uniform biofilm growth in 10% FBS as shown in  FIG. 22 . 
       FIG. 23  is a microscopy image of a SAW biofilm sensor assembled in accordance with  FIGS. 2-7E  after biofilm growth experiments in LB media; 
       FIG. 24  is a microscopy image of the SAW biofilm sensor of  FIG. 23  assembled in accordance with  FIGS. 2-7F , after biofilm growth experiments in 10% FBS; 
     After biofilm growth experiments in each media, the presence of bacterial biofilm on the SAW sensor was confirmed by optical microscopy as shown images in  FIGS. 23 and 24 . 
     As shown by the data, the 45 nm ALD Al 2 O 3  passivated SAW sensor was able to measure biofilm growth repeatably using oxygen plasma cleaning between experiments. The final frequency shift results in LB media were more variable than those in 10% FBS since the composition of the LB media was a more favorable environment to  E. coli , thereby indicating non-uniform biofilm growth in each experiment [47]. The observed SAW sensor outputs in both media followed the same growth trends. Moreover, the 10% FBS results suggest that the SAW sensor can be applied to in vivo biofilm detection in the future. Since the FBS is composed of blood proteins and plasma, the serum can be used to mimic an in vivo environment. The resonant frequency shift results of the SAW sensor in 10% HIS are more repeatable than results in LB media, rendering reliable operation of the sensor in an in vivo environment more likely. The effective passivation of the sensor using an ALD Al 2 O 3  film also contributed to reliable sensing in 10% FBS. 
       FIG. 25  is a microscopy image of a SAW biofilm sensor, such as SAW biofilm sensor  200  assembled in accordance with  FIGS. 2-7E  after cleaning with deionized water. 
       FIG. 26  is a microscopy image of the SAW biofilm sensor of  FIG. 25  after ultrasonication cleaning in acetone for 3 hours. 
       FIG. 27  is a microscopy image of the SAW biofilm sensor of  FIGS. 25 and 26  after oxygen plasma cleaning for 30 seconds. 
       FIG. 28  is a microscopy image of the SAW biofilm sensor of  FIGS. 25, 26 and 27  after a second biofilm growth experiment and oxygen plasma cleaning for 30 seconds. 
     The results indicated by  FIGS. 25 to 28  illustrate that the SAW sensor  200  can be reused after oxygen plasma cleaning, allowing for consecutive biofilm formation experiments using one sensor. 
     In view of the foregoing description, those skilled in the art will understand and appreciate that a novel ALD Al 2 O 3  film passivated SAW sensor for real time biofilm monitoring has been successfully demonstrated. A high quality c-axis oriented ZnO film was deposited by PLD, and the sensor was effectively passivated by 45 nm of Al 2 O 3  film using ALD to prevent ZnO damage in the bacterial growth media and animal serum. For the reliable passivation of the ZnO SAW sensor, ALD was an important fabrication method based on its highly dense and conformal film deposition capabilities. The SAW sensor can be reused after oxygen plasma cleaning, allowing for consecutive biofilm formation experiments using one sensor. The detection limit of the SAW sensor was approximately 5.3 pg. The resonant frequency shift results of the SAW sensor followed natural bacterial biofilm growth properties not only in LB media which provided a favorable bacterial growth environment, but also in 10% FBS as a simulated in vivo environment. These results validate the application of the SAW sensor for real-time bacterial growth monitoring. 
       FIG. 29  is a perspective view a top perspective view of an exemplary embodiment of a SAW biofilm sensor  600  disposed on a substrate  610 . The SAW biofilm sensor  600  includes an IDT  602  and an RF reflective tag  604  each disposed on the substrate  610  on an opposite side of a biofilm  40  such that a transmitted SAW  614  originating from the IDT  602  is reflected by the reflective tag  604  as a reflected SAW  616  back to the IDT  602 . The IDT  602  senses the reflected SAW  616  and via an antenna  606  in RF coupling  612  to an RF reader  608  to enable remote biofilm growth detection. 
     This SAW sensor  600  combined with RF wireless communication techniques can be used to detect in vivo biofilm growth, which is the groundwork for developing an implantable sensor for early biofilm detection and prevention of major infections. 
     Although the present disclosure has been described in considerable detail with reference to certain preferred version thereof, other versions are possible and contemplated. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred versions contained therein. 
     Any element in a claim that does not explicitly state “means for” performing a specified function or “step for” performing a specified function, is not to be interpreted as a “means” or “step” clause as specified in 35 U.S.C. §112, ¶6. In particular, the use of “step of” in the claims is not intended to invoke the provisions of 35 U.S.C. §112, ¶6. 
     While several embodiments of the disclosure have been shown in the drawings, it is not intended that the disclosure be limited thereto, as it is intended that the disclosure be as broad in scope as the art will allow and that the specification be read likewise. Therefore, the above description should not be construed as limiting, but merely as exemplifications of particular embodiments. Those skilled in the art will envision other modifications within the scope of the claims appended hereto. 
     In the claims, any reference signs placed between parentheses shall not be construed as limiting the claim. The word “comprising” does not exclude the presence of elements or steps other than those listed in a claim. The word “a” or “an” preceding an element does not exclude the presence of a plurality of such elements. The embodiments of the present disclosure may be implemented by means of hardware comprising several distinct elements, and/or by means of a suitably programmed processor. In the device claim enumerating several means, several of these means may be embodied by one and the same item of hardware. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. 
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